Tusc2 therapies

ABSTRACT

A method for predicting a subject&#39;s response to a TUSC2 therapy is provided. In particular, a subject&#39;s response is predicted based on the proportion of cancers cells that are apoptotic. Also provided is a method of treating a subject previously predicted to have a favorable response with a TUSC2 therapy. Methods for treating cancer by administration of a TUSC2 therapeutic in conjunction with an EGFR inhibitor and/or a protein kinase inhibitor are also disclosed. Kits and reagents for use in TUSC2 therapy are provided.

This application is a continuation of U.S. application Ser. No.14/480,341, filed Sep. 8, 2014, which is a divisional of U.S.application Ser. No. 13/410,811, filed Mar. 2, 2012, which claims thebenefit of U.S. Provisional Patent Application No. 61/448,463, filedMar. 2, 2011; 61/472,530, filed Apr. 6, 2011; 61/513,244, filed Jul. 29,2011; and 61/603,686, filed Feb. 27, 2012, each of which is incorporatedherein by reference in its entirety.

This invention was made with Government support under grant nos.CA-016672, CA-070907 and CA-113450 awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present embodiments provided herein relate generally to the fieldsof molecular biology and cancer therapies.

2. Description of Related Art

As the molecular and genetic mechanisms of oncogenesis become betterelucidated, the focus of cancer therapy has shifted from the tissue tothe genetic level (Bishop, 1991). Mutations in two major classes ofgenes, oncogenes and tumor suppressor genes (TSGs), play central rolesin the oncogenic process. TSGs appear to require homozygous deletion ormutation for inactivation, and restoration of TSG expression is feasiblein human tumors (Lowe et al., 2004; Roth, 2006). Intratumoral injectionof retroviral or adenoviral vectors expressing the wildtype TSG p53 havebeen performed in patients with locally advanced non-small cell lungcancer and head and neck cancer (Swisher et al., 1999; Roth et al.,1996; Clayman et al., 1998). These studies have demonstrated that viralvectors expressing the TSG p53 can be safely injected into tumorsrepetitively and can mediate tumor regression. However, because of thesystemic immune response, current viral vectors are limited tointratumoral administration, which does not have an effect on tumormetastases, the primary cause of cancer-related death. Thus developmentof therapies for intravenous, systemic TSG replacement would represent asignificant advance.

Homozygous deletions in the 3p21.3 region in lung cancer cell lines andprimary lung tumors have lead to the identification of multiple geneswith tumor suppressor activity from this region (Lerman et al., 2000).

SUMMARY OF THE INVENTION

In a first embodiment, there is provided a method for predicting aresponse to a TUSC2 (also known as FUS1) therapy in a subject having acancer, wherein the subject is being evaluated as a candidate for TUSC2therapy, comprising assessing apoptosis in cancer cells of the subject,wherein if 10% or more of the cancer cells are apoptotic, then thesubject is predicted to have a favorable response to TUSC2 therapy. Forexample, in certain aspects, the subject is predicted to have afavorable response to a TUSC2 therapy if at least 20%, 25%, 30%, 35%,40%, 45%, 50%, 60% or more of the cancer cells are apoptotic.Conversely, in certain aspects, if fewer than 10% of the cancer cellsthe subject are apoptotic (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, orless), the subject is predicted to have a poor response to the TUSC2therapy. For example, a favorable response to TUSC2 therapy can comprisea reduction in tumor size or burden, blocking of tumor growth, reductionin tumor-associated pain, reduction in cancer associated pathology,reduction in cancer associated symptoms, cancer non-progression,increased disease free interval, increased time to progression,induction of remission, reduction of metastasis, or increased patientsurvival.

In a further embodiment there is provided a method of selecting asubject having a cancer for a TUSC2 therapy comprising assessingapoptosis in cancer cells of the subject, wherein if 10% or more of thecancer cells are apoptotic, then the subject is selected for the TUSC2therapy. For example, in certain aspects, the subject is selected for aTUSC2 therapy if at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60% or moreof the cancer cells are apoptotic. On the other hand, if fewer than 10%of the cancer cells the subject are apoptotic (e.g., 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, or less), the subject is not selected for the TUSC2therapy.

In certain embodiments, assessing apoptosis in cancer cells or a sampleof cancer cells comprises testing the cells for a marker of apoptosis. Avariety of apoptotic markers are known in the art and can be used toassess apoptosis in cancer cells. For example, apoptosis can be assessedby testing for caspase activation, membrane blebbing, loss ofmitochondrial membrane integrity, or DNA fragmentation. Varioustechniques may be used for testing cells to assess apoptosis and thetesting method will depend upon the marker that is being used. Forexample, testing for apoptosis may comprise performing an ELISA, animmunoassay, a radioimmunoassay (MA), an immunoradiometric assay, afluoroimmunoassay, a chemiluminescent assay, a bioluminescent assay, agel electrophoresis, a Western blot analysis, a southern blot, flowcytometry, in situ hybridization, positron emission tomography (PET),single photon emission computed tomography (SPECT) imaging or amicroscopic assay. Thus, in certain aspects, cancer cells are tested foran apoptotic marker in vivo (e.g., by PET or SPECT imaging).

In certain embodiments, testing cells for a marker of apoptosiscomprises contacting the cancer cells with a reagent that labels cellscomprising a marker of apoptosis. Examples of reagents that can be usedto label apoptotic cells include, but are not limited to, antibodies,small molecules, stains, enzymes nucleic acid probes and aptamers. Forinstance, in certain cases, apoptosis may be assessed by detecting DNAfragmentation, such as by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling (TUNEL). In this case a subjectwould be predicted to have a favorable response to a TUSC2 therapy, if10% or more of the cancer cells in a sample from the patient are TUNELpositive.

The types of cancer cell samples that are assessed for apoptosis willdepend upon the type of cancer involved. For example, in the case of acancer that presents as one or more solid tumor, the sample may be tumorbiopsy sample from a primary cancer site or a metastatic site. Cancercells may also be comprised in other body samples, such as, serum,stool, urine and sputum. In certain aspects, wherein a sample comprisesa large number of non-cancer cells, assessing cancer cells for apoptosismay additionally comprise identifying the cancer cells and assessing theidentified cancer cells for apoptosis.

In a further embodiment, there is provided a method for treating asubject having a cancer, wherein it was previously determined (orpreviously estimated) that at least 10% of the cells of said cancer areapoptotic, the method comprising administering a TUSC2 therapy to thesubject. For example, in certain aspects, at least 20%, 25%, 30%, 35%,40%, 45%, 50%, 60% or more of the cancer cells of the subject werepreviously determined to be apoptotic. As used herein a TUSC2 therapycan be any type of therapy that provides or causes expression of a TUSC2polypeptide in a cancer cell (see, e.g., U.S. Pat. No. 7,902,441,incorporated herein by reference). For example, a TUSC2 therapy maycomprise delivery of a TUSC2 polypeptide or TUSC2 expression vector tocancer cell. A therapy may, for instance, be delivered viananoparticles, or in the case of nucleic acid expression vectors,through the use of a viral vector.

In certain embodiments, administration of a TUSC2 therapy comprisesadministration of a TUSC2 expression vector, such a DNA plasmid encodingTUSC2. An expression vector for use according to the embodimentsprovided herein will generally comprise control elements for theexpression of the TUSC2 coding sequence. For example, a vector cancomprise a promoter and enhancer element that are effective forexpression in cancer cell of interest. In certain aspects, for instance,TUSC2 expression is provided by a CMV promoter or recombinant versionthereof, such as the CMV promoter construct described in U.S. PatentPubln. No. 20070092968, incorporated herein by reference. In certainembodiments, a vector provided herein comprises a modified CMV promoter.In certain embodiments, a vector provided herein comprises a mini-CMVpromoter. Additional expression control elements can be included suchas, for example, an intron, a drug response element, a RNA stabilizingor destabilizing sequence, a cellular localization signal, apolyadenylation signal sequence and/or an optimized translation startcodon. Plasmid DNA vectors may also comprise sequences that helpfacilitate DNA production, such as, a bacterial origin of replicationand/or a drug resistance marker. In certain specific aspects, the TUSC2expression vector is the pLJ143/KGB2/FUS1 plasmid (SEQ ID NO: 1).

Methods for delivery of an expression vector to cells (e.g., in vivodelivery) are well known in the art and include, without limitation,nanoparticles (e.g., liposome nanoparticles), lipid conjugates and viralvectors. In certain aspects, a TUSC2 expression vector is administeredin a nanoparticle, such asN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride(DOTAP):cholesterol liposome nanoparticle. A skilled artisan willrecognize that various properties of liposomes can be adjusted tooptimize vector delivery. For example, the liposomes may be adjusted tohave a certain size range and/or a particular ratio of DNA to lipid; DNAto cholesterol; or lipid to cholesterol. For instance, in the case of aDOTAP:cholesterol liposome, the DOTAP:cholesterol ratio can be definedas between about 1.5:1 and 1:1.5, such as about 10:9. In furtheraspects, a TUSC2 expression vector is provided in a liposomenanoparticle, wherein the nanoparticles comprise an average particlesize of between about 50 and about 500 nm (e.g., 200-500 nm). In stillfurther aspects, a TUSC2-nanoparticle formulation can be defined bytheir optical density (OD), such as having OD₄₀₀ of between about 0.65and 0.95.

In still further embodiments a TUSC2 therapy can comprise administrationof a TUSC2 polypeptide. Methods for administration of TUSC2 polypeptideare described for example in U.S. Publn. Nos. 20060251726 and20090023207, incorporated herein by reference. A TUSC2 polypeptide maybe modified to enhance its activity and/or ability to enter cancercells. For instance, the polypeptide can be modified with a lipid moiety(e.g., myristoylated). In certain aspects a TUSC2 in provided as ananoparticle (e.g., a lipid-based nanoparticle) such as, asuperparamagnetic nanoparticle, a nanoshell, a semiconductornanocrystal, a quantum dot, a polymer-based nanoparticle, asilicon-based nanoparticle, a silica-based nanoparticle, a metal-basednanoparticle, a fullerene or a nanotube.

A TUSC2 therapy according to the embodiments provided herein istypically formulated in a pharmaceutically acceptable carrier. Such atherapy may be delivered, for example, intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally, viainhalation (e.g. aerosol inhalation), by injection or by infusion, andthe route of delivery can depend upon the type of cancer to be treated.For example, a TUSC2 expression vector complexed with DOTAP:cholesterolliposome can be administer via intravenous infusion. In certain specificaspects, a TUSC2 therapy is administered intravenously in a dose of fromabout 0.01 mg/kg to about 0.10 mg/kg, such as a dose of about 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08 0.09 or 0.10 mg/kg. In furtheraspects, a TUSC2 therapy, can be administer two or more times (e.g., 3,4, 5, 6, 7, 8, 9 or 10 times). The timing between doses of such atherapy can be varied and can include, without limitation, about 1, 2 or3 days, about 1, 2, or 3 weeks or 1 month or more between doses.

In yet a further embodiment, there is provided a method for treating asubject having a cancer, comprising administering a TUSC2 therapy to thesubject in conjunction with one or more anti-inflammatory agent. Forexample, the anti-inflammatory agent may be administered before, afteror during a TUSC2 therapy. In a further aspects, more than oneanti-inflammatory agent is administered, such as administration of anantihistamine and a corticosteroid. Thus, in certain specific aspectsthe anti-inflammatory for use in conjunction with a TUSC2 therapy isdiphenhydramine and/or dexamethasone.

In certain embodiments, a cancer for treatment or assessment may presentas a tumor, such as primary or metastatic tumor. A cancer may be anearly stage cancer, or may be a metastatic or late stage cancer. Incertain aspects, the cancer is an oral cancer, oropharyngeal cancer,nasopharyngeal cancer, respiratory cancer, a urogenital cancer, agastrointestinal cancer, a central or peripheral nervous system tissuecancer, an endocrine or neuroendocrine cancer, a hematopoietic cancer, aglioma, a sarcoma, a carcinoma, a lymphoma, a melanoma, a fibroma, ameningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer,renal cancer, biliary cancer, prostatic cancer, pheochromocytoma,pancreatic islet cell cancer, a Li-Fraumeni tumor, thyroid cancer,parathyroid cancer, pituitary tumors, adrenal gland tumors, osteogenicsarcoma tumors, multiple neuroendrcine type I and type II tumors, breastcancer, lung cancer (e.g., a non-small cell lung cancer (NSCLC) or smallcell lung cancer (SCLC)), head & neck cancer, prostate cancer,esophageal cancer, tracheal cancer, skin cancer brain cancer, livercancer, bladder cancer, stomach cancer, pancreatic cancer, ovariancancer, uterine cancer, cervical cancer, testicular cancer, coloncancer, rectal cancer or skin cancer. In further aspects a cancer may bedefined as a cancer that is resistant to one or more anticancer therapy,such a chemotherapy resistant cancer. For example, the cancer may be acancer that is resistant to a platinum-based chemotherapeutic, such ascisplatin.

In further embodiments, a method provided herein further comprisesadministering at least a second anticancer therapy. For example, amethod can comprise treating a subject having a cancer, wherein it waspreviously determined that at least 10% of the cells of said cancer areapoptotic, comprising administering a TUSC2 therapy and at least asecond anticancer agent to the subject. The second anticancer therapymay be, without limitation, a surgical therapy, chemotherapy (e.g.,administration of a protein kinase inhibitor or a EGFR-targetedtherapy), radiation therapy, cryotherapy, hyperthermia treatment,phototherapy, radioablation therapy, hormonal therapy, immunotherapy,small molecule therapy, receptor kinase inhibitor therapy,anti-angiogenic therapy, cytokine therapy or a biological therapies suchas monoclonal antibodies, siRNA, antisense oligonucleotides, ribozymesor gene therapy. Without limitation the biological therapy may be a genetherapy, such as tumor suppressor gene therapy, a cell death proteingene therapy, a cell cycle regulator gene therapy, a cytokine genetherapy, a toxin gene therapy, an immunogene therapy, a suicide genetherapy, a prodrug gene therapy, an anti-cellular proliferation genetherapy, an enzyme gene therapy, or an anti-angiogenic factor genetherapy.

In still a further embodiments provided herein is a kit comprising aTUSC2 therapeutic. For example, in some aspects, a kit provided hereincomprises a TUSC2 therapeutic and a reagent for testing cells for amarker of apoptosis, such as a TUNEL reagent. In further aspects, a kitcomprises a TUSC2 therapeutic and one or more anti-inflammatory agents.In still further aspects the kit may comprise one more additionalcomponents including, but not limited to, a reagent for assessingapoptosis in a cell sample, an anti-inflammatory agent, pharmaceuticallyacceptable dilution agent, a syringe, an infusion bag, an infusion line,and/or a set of instruction for use of the kit.

In yet a further embodiment provided herein are compositions, therapies,and methods for treating a subject having a cancer, comprisingadministering to the subject a TUSC2 therapy (e.g., a TUSC2 polypeptideor a TUSC2 expression vector) in conjunction with a second anticanceragent, such as a chemotherapeutic. For example, the chemotherapeutic canbe a protein kinase inhibitor, such as a Src or Akt kinase inhibitor. Insome aspects, the chemotherapeutic is a epidermal growth factor receptor(EGFR) inhibitor.

In certain embodiments, a method is provided for treating a subjecthaving a cancer, comprising administering to the subject a TUSC2 therapyin conjunction with a protein kinase inhibitor. For instance, the TUSC2therapy can be administered, before, after or essentially concomitantlywith the protein kinase inhibitor. Thus, in some embodiments, acomposition is provided comprising a TUSC2 therapeutic and a proteinkinase inhibitor inhibitor in a therapeutically effective amount totreat a cancer. Protein kinase inhibitors for use according to theembodiments include, without limitation, EGFR, VEGFR, AKT, Erb1, Erb2,ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR,c-Kit, eph receptor or BRAF inhibitors. For example, the protein kinaseinhibitor can be Afatinib, Axitinib, Bevacizumab, Bosutinib, Cetuximab,Crizotinib, Dasatinib, Erlotinib, Fostamatinib, Gefitinib, Imatinib,Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Panitumumab, Pazopanib,Pegaptanib, Ranibizumab, Ruxolitinib, Saracatinib, Sorafenib, Sunitinib,Trastuzumab, Vandetanib, AP23451, Vemurafenib, CAL101, PX-866, LY294002,rapamycin, temsirolimus, everolimus, ridaforolimus, Alvocidib,Genistein, Selumetinib, AZD-6244, Vatalanib, P1446A-05, AG-024322,ZD1839, P276-00, GW572016, or a mixture thereof. In certain aspects, theprotein kinase inhibitor is an AKT inhibitor (e.g., MK-2206, GSK690693,A-443654, VQD-002, Miltefosine or Perifosine).

EGFR-targeted therapies for use in accordance with the embodimentsinclude, but are not limited to, inhibitors of EGFR/ErbB1/HER,ErbB2/Neu/HER2, ErbB3/HER3, and/or ErbB4/HER4. A wide range of suchinhibitors are known and include, without limitation, tyrosine kinaseinhibitors active against the receptor(s) and EGFR-binding antibodies oraptamers. For instance, the EGFR inhibitor can be gefitinib, erlotinib,cetuximab, matuzumab, panitumumab, AEE788; CI-1033, HKI-272, HKI-357 orEKB-569. In certain embodiments, the compositions and therapies providedherein are administered systemically or locally. In one embodiment, thecompositions and therapies provided herein are administeredsystemically. In certain aspects, an EGFR inhibitor is administered to apatient before, after or essentially concomitantly with a TUSC2 therapy.For example, the therapies may be co-administered, such as byco-administration in an intravenous infusion. In certain embodiments,TUSC2 and EGFR inhibitors can be administered in any amount effective totreat cancers. In certain embodiments, the compositions, therapies, andmethods provided herein comprise administering TUSC2 and EGFR inhibitorsin lower doses than either composition administered alone. In certainembodiments, the compositions, therapies, and methods compriseadministering TUSC2 and EGFR inhibitors in lower doses that reduce sideeffects. In certain embodiments, the compositions, therapies, andmethods comprise administering TUSC2 and EGFR inhibitors in doseseffective to provide additive, cooperative, or synergistic effect thanthat provided by either composition administered alone. In certainaspects, cancers for treatment with such therapies can be any of thosedescribed herein, such as lung cancers (e.g., non-small cell lungcancer). In certain preferred aspects, a cancer for treatment with acombination therapy is an EGFR-expressing cancer. In certainembodiments, the EGFR-expressing cancer comprises at least 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% tumor cells expressing EGFR.

In yet still a further embodiment provided herein is a method fortreating a subject having a cancer, wherein it was previously determinedthat the cancer expresses an EGFR, the method comprising administeringto the subject a TUSC2 therapy in conjunction with a EGFR inhibitor. Incertain embodiments, provided herein is a method for treating a subjecthaving a cancer comprising the step of determining whether the cancerexpresses an EGFR, and administering to the subject a TUSC2 and an EGFRinhibitor. Methods for assessing the EGFR-expression status of a cancerhave been described, for example in U.S. Patent Publn. No. 20110052570,incorporated herein by reference. In certain aspects, theEGFR-expressing cancer can be a cancer that expresses a mutant EGFR,such as a cancer expressing an EGFR having a L858R and/or T790Mmutation. In certain embodiments, the compositions and therapiesprovided herein are administered to the patient that have anEGFR-expressing cancer that comprises at least 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% tumor cells expressing EGFR. In still furtheraspects, the subject for treatment has a cancer that was previouslydetermined to express an EGFR and in which at least 10% of the cells ofthe cancer are apoptotic. In certain embodiments, the methods providedherein further comprise determining whether at least 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% of the cells of the EGFR-expressing cancer areapoptotic.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein. Likewise, aspects of the present embodiments discussed in thecontext of a method for treating a subject are equally applicable to amethod of predicting response in a subject and vise versa.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Certain aspects of the embodiments concern selecting a subject having acancer for a TUSC2 therapy or for predicting a response to a TUSC2therapy in a subject. In this context, “a poor response” to a TUSC2therapy means that administration of a TUSC2 therapy, either alone or incombination with a further anticancer agent, is predicted to result inno significant treatment of a cancer (e.g., as measured by reduction oftumor mass, number of metastases, or rate of cancer cell proliferation)or symptoms of a cancer. On the other hand, “a favorable response” meansthat administration of a TUSC2 therapy, either alone or in combinationwith a further anticancer agent, is predicted to result in significanttreatment of a cancer (e.g., as measured by reduction of tumor mass,number of metastases, or rate of cancer cell proliferation) or cancersymptoms. For example, a favorable response can be a significantlyincreased period of relapse-free remission in a subject.

Other objects, features and advantages of the present embodiments willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments provided herein, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the present embodimentswill become apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentembodiments. The present embodiments may be better understood byreference to one or more of these drawings in combination with thedetailed description of specific embodiments presented herein.

FIG. 1A-C: FIG. 1A, A schematic representation of a the TUSC2 expressionvector pLJ143/KGB2/FUS1 (SEQ ID NO: 1). FIG. 1B, Change in apoptosispathway mRNAs analyzed in pre- and post-treatment biopsy specimens frompatient 31 using SA Apoptosis Signaling Nano-scale PCR Array. Genes inpost-treatment samples that differed from pretreatment controls by >3fold are shown as a scatter plot of log 10 post-treatment values vs log10 pretreatment values. Factors for which mRNA expressionincreased >3-fold post treatment appear above the line, while those thatdecreased by >3 fold appear below the line. Exogenous TUSC2 mRNAexpression was detected in the post-treatment biopsy from this patient.FIG. 1C, Canonical apoptosis pathway gene expression pertubationsfollowing TUSC2-nanoparticle treatment as detected by SA PRC Array andIPA Analysis. Molecules are represented as nodes, and the biologicalrelationship between two nodes is represented as an edge (Line). Theasterisks indicate up- (single asterisk) or down- (double asterisk)regulation. Nodes are displayed using various shapes that represent thefunctional class of the gene products. Edges are displayed with variouslabels that describe the nature of the relationship between the nodes(e.g., P for phosphorylation, T for transcription). The identified nodesindicate perturbation of elements of the intrinsic and extrinsicapoptotic pathways following treatment with DOTAP:chol-TUSC2.

FIG. 2A-B: FIG. 2A, In situ Proximity Ligation Assay (PLA) for TUSC2protein in tumor biopsies. A synthetic oligopeptide (GASGSKARGLWPFASAA;SEQ ID NO: 2) derived from the N-terminal amino-acid sequence of theTUSC2 protein was used to develop the anti-TUSC2 polyclonal antibody inrabbits used in this study. Red denotes TUSC2 positivity. DAPI nuclearstaining is blue. All panels represent overlays of TUSC2 antibody andDAPI staining. Detailed methods are provided in the SupplementaryMethods. Pre- and post-treatment biopsies from patients 13, 26, and 31were tested. Magnification is X40. Panels: (1) anti-TUSC2 antibody; (2)anti-TUSC2 antibody pre-absorbed with non-specific control peptide(NSP); (3) anti-TUSC2 antibody pre-absorbed with TUSC2 peptide (FP); (4)non-specific control antibody; (5) hematoxylin and eosin. FIG. 2B,Quantitation of PLA signals for pre- and post-treatment samples frompatients 13, 26, and 31. The anti-TUSC2 antibody was tested under theconditions described in A). The upper panels show PLA signals from therespective patient biopsies as detected by the anti-TUSC2 antibody with400× magnification. The lower panel presents quantitative comparisons ofsix independent fields from each biopsy treated under the specifiedconditions. TUSC2 expression was significantly increased inpost-treatment samples compared to pretreatment samples. TUSC2expression was not significantly altered by anti-TUSC2 antibodypre-absorption with non-specific control peptide (NSP), but wassignificantly decreased by pre-absorption with TUSC2 peptide (FP). *p<0.05 compared to corresponding pretreatment sample; ▪ p<0.05 comparedto post-treatment samples unabsorbed or pre-absorbed with NSP. Allcomparisons are by two-tailed unpaired Student's t-test assuming equalvariances as determined by F test.

FIG. 3A-B: DOTAP:chol-TUSC2 metabolic tumor response in a metastaticlung cancer patient. The patient is a 54 year old female with a largecell neuroendocrine carcinoma. She had received six prior chemotherapyregimens. Prior to entry in the protocol, two hepatic metastases wereprogressing on gemcitabine. The patient also had a metastasis in thehead of the pancreas and a peripancreatic lymph node (indicated byarrows). FIG. 3A, Pretreatment PET scan. The dose of Fluorodeoxyglucose(¹⁸F) was 8.8 mCi. FIG. 3B, Post-treatment PET scan performed 20 daysfollowing the fourth dose of DOTAP:chol-TUSC2. The dose ofFluorodeoxyglucose (¹⁸F) was 9.0 mCi. All scans were performed within a60 to 90 minute window after injection.

FIG. 4: TUSC2 expression was determined by immunohistochemistry. Thedashed line indicates the level of TUSC2 expression in a biopsy ofnormal bronchial epithelium from one patient. The asterisks indicatepatients who showed stable disease or minor response following treatmentwith DOTAP:chol-TUSC2 nanoparticles. No associations between the IHCmarker with treatment outcome was observed.

FIG. 5: Apoptotic index was determined by TUNEL staining. The asterisksindicate patients who showed stable disease or minor response followingtreatment with DOTAP:chol-TUSC2 nanoparticles. A maximum pretreatmentapoptotic index of greater than 10% was associated with stable diseaseor minor response following treatment with DOTAP:chol-TUSC2nanoparticles.

FIG. 6: Fus1 and Erlotinib combined treatment effect on colony formationof H1299 cells. Graph shows the results of colony formation assays aschange in total colony area relative to control for each treatmentcondition. “EV” indicates empty vector; Fus1 indicates a vectorcontaining Fus1; numerical values following “erlo” indicate μg ofErlotinib; PBS indicates Phosphate-Buffer Saline control.

FIG. 7: Fus1 and Erlotinib combined treatment effect on colony formationof H322 cells. Graph shows the results of colony formation assays aschange in total colony area relative to control for each treatmentcondition. “EV” indicates empty vector; Fus1 indicates a vectorcontaining Fus1; numerical values following “+” indicate μg ofErlotinib; PBS indicates Phosphate-Buffer Saline control.

FIG. 8: Fus1 and Erlotinib combined treatment effect on colony formationof A549 cells. Graph shows the results of colony formation assays aschange in total colony area relative to control for each treatmentcondition. “EV” indicates empty vector; Fus1 indicates a vectorcontaining Fus1; numerical values following “+” indicate μg ofErlotinib; PBS indicates Phosphate-Buffer Saline control.

FIG. 9: Fus1 and Erlotinib combined treatment effect on colony formationof H460 cells. Graph shows the results of colony formation assays aschange in total colony area relative to control for each treatmentcondition. “EV” indicates empty vector; Fus1 indicates a vectorcontaining Fus1; numerical values following “+” indicate μg ofErlotinib; PBS indicates Phosphate-Buffer Saline control.

FIG. 10: Fus1 and Erlotinib combined treatment effect on colonyformation of H1975 cells (H1975 cells have two EGFR mutations,L858R/T790M). Graph shows the results of colony formation assays aschange in total colony area relative to control for each treatmentcondition. “EV” indicates empty vector; Fus1 indicates a vectorcontaining Fus1; numerical values following “+” indicate μg ofErlotinib; PBS indicates Phosphate-Buffer Saline control.

FIG. 11A-B: FACS analysis was used to measure intracellular levels ofTNF-a, IL-15, IL-6, IL1b, IFNg, and IL-8 in peripheral blood monocytesand lymphocytes in pretreatment and posttreatment samples 24 hours afteradministration of the DOTAP:chol-TUSC2. For one patient peripheral bloodmononuclear cells (PBMC) were obtained 14 months following 12 treatments(Post 2). Only IL-15 showed detectable levels in lymphocytes andmonocytes. No statistically significant increases in the post-treatmentsamples were observed for any cytokine. All comparisons are bytwo-tailed paired Student's t-test. FIG. 11A, are results fromperipheral blood monocytes (Mo). FIG. 11B, are results from peripheralblood lymphocytes (Ly).

FIG. 12A-D: Effects of combination treatment of FUS1 and gefitinib(“Gef” or “G”) and erlotinib (“Erl” or “E”) on tumor cell growth and PTKactivities in NSCLC cells in vitro and in vivo. FIG. 12A, Effects oninduction of apoptosis using TUNEL reaction by FACS. FIG. 12B, Effectsof FUS1 and erlotinib on tumor cell growth in resistant H322, H1299.FIG. 12C, Evaluation of therapeutic efficacy and induction of apoptosisby systemic injection of FUS1 nanoparticles and oral administration ofgefitinib in human H322 orthotopic lung tumors in nude mice. Freshfrozen tumors were stained for apoptosis by in situ TUNEL staining. FIG.12D, Effects on EGFR, AKT, and ERK activities by western blot analysis.

FIG. 13: FUS1 nanoparticle and Erlotinib combination therapy on A549Lung Colonies. Mice (5-6 wk old nu/nu) were injected in the tail veinwith 10⁶ A549 cells. Ten days later treatment was begun with erlotinib30 mg/kg orally daily for 7 days and FUS1 nanoparticles (25 μg)intravenously on days 10, 13, and 16. Mice were killed on day 36 andlung tumors counted. Erlo=erlotinib; EV=empty vector. The FUS1+erlotinibgroup is significantly less than all other groups by the two independentsample Wilcoxon rank sum test (p<0.0005).

FIG. 14A-C: Effect of Conditioned Medium (CM) from FUS1-nanoparticleTreated H1299 Cells on H1299 Tumor Cell Growth (FIG. 14A) and apoptosis(FIG. 14B). FIG. 14C, Protein profiles of CMs on ProteinChip Array bySELDI-MS.

FIG. 15A-B: Bystander effects induced by FUS1-nanoparticle-mediated genetransfer in NSCLC H1299 cells by FACS analysis (FIG. 15A and FIG. 15B).The populations of dead/apoptotic cells are represented by both PI(upper left quadrant)-positive and PI/GFP (“R+G”) positive cells.FUS1-transfected H1299 cells were used as effecter cells andAd-GFP-transduced H1299 cells as target cells and mixed at a ratio of1:1.

FIG. 16: Effect of FUS1 nanoparticles alone on lung cancer cell lines.Top panel is a representation of Western blot to detect FUS1 expressionin cancer cell lines HCC366, H322, A549 or H2887. β-actin a was used asa loading control. Bottom panel are graphs that show the total number ofviable cells for each of the four cell lines calculated at 24, 48 and 72hours upon treatment with FUS1 nanoparticles or empty vector (EV).

FIG. 17: Single drug treatment of MK2206 on lung cancer cells. Graphshows the inhibitory concentration 50 (IC₅₀) for AKT inhibitor MK2206 onvarious cancer cells. Cell lines that were further analyzed areindicated by arrows.

FIG. 18: FUS1/MK2206-induced cell death in lung cancer cell lines.Graphs show the relative survival rates for the indicated cancer cells(y-axis) when contacted with empty vector (EV) or FUS1 nanoparticles inthe presence of increasing concentrations of MK2206 (x-axis).

FIG. 19: FUS1/MK2206 inhibit colony formation in lung cancer cell lines.Graphs show the relative percent of colony formation by the indicatedcells upon treatment with empty vector (EV); FUS1 nanoparticles (FUS1);MK2206; empty vector+MK2206 (EV+MK); or FUS1 nanoparticles+MK2206(FUS1+MK). * indicates a statistically significant difference in theamount of colony formation between the two treatments.

FIG. 20: FUS1/MK2206 induced apoptosis in lung cancer cell lines. Theeffects of empty vector (EV); FUS1 nanoparticles (FUS1); MK2206; emptyvector+MK2206 (EV+MK2206); or FUS1 nanoparticles+MK2206 (FUS1+MK2206) onthe cell cycle were examined in the four indicated cancer cell line.Treated cells were stained by propidium iodide (PI) and analyzed by flowcytometry. Histograms show cell count (y-axis) versus PI intensity as ameasure of DNA content. The horizontal bar in each histogram indicatesapoptotic cells as assessed by PI staining of DNA.

FIG. 21: Immunoblot of p-AKT, p-AMPK and p-mTOR in FUS1/MK2206-treatedcell lines. Phosphorylation specific antibodies were used to assessexpression of phosphorylated AMPK, AKT, mTOR and S6K in HCC366 or H322cells. Cells were treated with empty vector (EV); FUS1 nanoparticles(FUS1); MK2206; empty vector+MK2206 (EV+MK); or FUS1nanoparticles+MK2206 (FUS1+MK) prior to assessment for phosphorylatedprotein expression. Immunoblot of β-actin was used as a loading control.

FIG. 22: The effect of AMP-activated protein kinase (AMPK)-specificsiRNA on FUS1/MK2206-induced cell death. Cell survival was assessed inHCC366 and H322 cells treated with FUS1 nanoparticles and variousconcentrations of MK2206 in the presence or absence of siRNA targeted toAMPK. Top panels are representations of Western blots confirmingeffective knock-down of AMPK expression upon introduction of siRNA.Bottom panels are graphs showing relative cell survival (y-axis) atvarious concentrations of MK2206 (x-axis) with and without AMPK siRNA asindicated.

FIG. 23: The effect of AMPK inhibitor on FUS1/MK2206-induced cell death.Cell survival was assessed in HCC366 and H322 cells treated with FUS1nanoparticles and various concentrations of MK2206 in the presence orabsence of AMPK inhibitor Compound C. Graphs show relative cell survival(y-axis) at various concentrations of MK2206 (x-axis) with and withoutAMPK inhibitor as indicated.

FIG. 24: Combination effects of FUS1 and MK2206 in H322 xenograft mousemodel. The chart shows the effects of empty vector (EV); FUS1nanoparticles (FUS1); MK2206; empty vector+MK2206 (EV+MK2206); or FUS1nanoparticles+MK2206 (FUS1+MK2206) on H322 tumor growth in vivo. Totaltumor volume (y-axis) is plotted as function of time (x-axis).Expression of FUS1 and activity of MK2206 (as evidenced by reduced p-AKTexpression) was histologically confirmed in samples from the mice.

FIG. 25: Proposed mechanism of FUS1/MK2206-induced cell death throughAKT/AMPK/mTOR pathway. Schematic shows example members of a signalingpathway modulated by FUS1 and MK2206 treatment.

FIG. 26A-B: Afatinib synergistically inhibits colony formation when usedin conjunction with TUSC2 nanoparticles. Results are shown for colonyformation assays in H1299 (FIG. 26A) or H322 (FIG. 26B) cells. TUSC2nanoparticle treatment is indicated by “FUS1” versus control treatment“301.” These treatments were applied in conjunction with controltreatment “CTR”; 0.5 or 1.0 μg of Erlotinib (“Erlo-0.5” or “Erlo-1.0”);or 0.5, 1.0 or 2.0 of Afatinib (“Afa-0.5”, “Afa-1.0” or “Afa-2.0”) asindicated.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The development of cancer involves the deregulation of number ofcellular pathways that control normal cell growth. Crucially, healthycells express a number of tumor suppressor genes, which act as moleculargatekeepers and prevent uncontrolled cell division. A necessary step inthe development of a cancer cells, therefore, is disruption of tumorsuppressor signaling pathways. In view of this, one promising avenue forcancer therapy involves expression of tumor suppressor genes in cancercells to restore normal cellular growth controls. Such therapies mayprove less toxic than standard radiation and chemotherapeutic regimes,as normal, noncancerous cells, naturally express the suppressor genes.

The studies detailed herein demonstrate the therapeutic delivery ofTUSC2 was safe, resulted in disease stabilization in a number of thestudy patients and shows promise for clinical effect. In the study,thirty-one human patients were treated at 6 dose levels ranging from0.01 to 0.09 milligrams per kilogram or DOTAP:chol-TUSC2. The therapyresulted increased expression of TUSC2 in post-treatment tumor specimensbut not in pretreatment specimens or peripheral blood lymphocytecontrols. Likewise, TUSC2 protein expression was effectively detected inpost-treatment tissues and expression was shown to alter the regulationof a number of genes involved in both the intrinsic and extrinsicapoptotic pathways (see, FIG. 1C). Five patients achieved achievingstable disease (2.6-10.8 months, including 2 minor responses). Onepatient with stable disease had a metabolic response on positronemission tomography (PET) imaging (FIG. 3). Thus, the studiesdemonstrate the safety of TUSC2 therapy and indicate that the therapymay be effective to improve patient outcome.

Owing to the fact that all cancer cells develop differently, one crucialimpediment to anticancer therapy is that certain cancers respond to agiven therapy while others prove recalcitrant. Therefore, in order toprovide effective therapy, methods are needed to identify subjects thatwill favorably respond to a given therapy. The studies detailed heredemonstrate for the first time effective methods to identify cancerpatients who are responders to a TUSC2 therapy regime. Counterintuitively, it has been demonstrated that cancers exhibiting a highproportion of apoptotic cells are more susceptible to therapy.Specifically, patients that respond favorably to the TUSC2 therapy hadcancers, wherein about 10% or more of the cells were identified asapoptotic by a TUNEL assay to detect DNA fragmentation (see, e.g., FIG.5). Accordingly, provided herein is a method for predicting whether asubject will have a favorable response to a TUSC2 therapy by testing thecancer cells of the subject to determine the proportion of the cellsthat are apoptotic. Likewise, methods for treating subjects who arepreviously determined to have a cancer with a high proportion (e.g., 10%or greater) of apoptotic cells are provided. These methods will allowfor identification and treatment of populations of cancer patients whowill likely response to TUSC2 therapies thereby improving the efficacyof the therapy.

Despite the relatively low toxicity exhibited by TUSC2 therapeutics,minor adverse responses were initially noted in the clinical studiesdescribed here. It was found, however, that such adverse reactions couldbe nearly completely ablated by the use of an anti-inflammatory regimein conjunction with the TUSC2 therapy. Specifically, the administrationof an antihistamine (diphenhydramine) and a corticosteroid(dexamethasone) immediately preceding and immediately following theTUSC2 administration protected patients from adverse reactions andallowed for higher doses of the TUSC2 therapeutic to be administered.This is an important finding given the possible need to provide higherdoses of the therapy for effective clinical benefit. Thus, a methods isprovided for treating a patient with a TUSC2 therapeutic comprisingadministering the therapy in conjunction with one or moreanti-inflammatory agent. Accordingly, the two therapies can be includedin combined therapeutic regime to increase anti-cancer efficacy.Likewise, when combined the dose of one or both therapies could bereduced while still maintaining effectiveness, thereby potentiallyreducing the side effects of the combined therapy. Such combined regimesmay also show particular effect in specific patient populations, such asthose having cancers that are EGFR positive, demonstrate increasedapoptotic activity and/or exhibit increased kinase (e.g., AKT) activity.

The embodiments and working examples provided herein demonstrate for thefirst time that TUSC2 therapies show increased effectiveness whencombined with EGFR-targeted therapies. Specifically, studies presentedhere show that erlotinib, an EGFR tyrosine kinase inhibitor, issignificantly more effective in reducing cancer cell growth (as measuredby colony formation) when applied in conjunction with a TUSC2therapeutic (see, e.g., FIGS. 6-10 and Tables 5-8). In all five cancercell lines that were tested application of TUSC2 nanoparticles was ableto sensitize the cancer cell to the effects of Erlotinib. The combinedtreatment was able to achieve a similar level of inhibition of colonyformation while using less than half of the amount of a erlotinib (1.0μg versus 2.3 μg) treatment when provided alone. Moreover, at the highererlotinib treatment levels (2.3 μg) the combination treatment farexceeded the amount of inhibition that was achievable with either agentalone. These results were confirmed using an in vivo murine tumorexplants model. Results shown in FIG. 13 demonstrate that thecombination of TUSC2 therapy and erlotinib significantly reduced thenumber of tumor nodules in the lungs of treated animals as compared toeither treatment alone. Further studies in four different cancer celllines confirmed that TUSC2 therapy likewise was able to sensitize cellsto killing by a second EGFR-targeted agent, gefitinib (FIG. 12).Moreover, the tyrosine kinase inhibitor afatinib, which likewise acts toinhibit EGFR signaling, synergistically inhibited cancer cell colonyformation when combined with TUSC2 treatment (FIG. 26A-B). Thus, TUSC2therapy can be used to sensitize cancer cells to the effects ofEGFR-targeted therapies (such as erlotinib, afatinib or gefitinib) andthereby reduce the effective amount of the EGFR-targeted therapyrequired for effective treatment of a cancer.

Yet further studies presented here demonstrate that TUSC2 therapeuticsare also able to sensitized cancer cells to the effects of proteinkinase inhibitors. For example, as shown in FIG. 18, the cancer cellkilling effect of AKT kinase inhibitor MK2206 were greatly increasedwhen the inhibitor was used in conjunction with TUSC2 nanoparticles.Interestingly, TUSC2 treatment was able to render cells that wereotherwise highly resistant to the AKT inhibitor (such as HCC366 cells)susceptible to MK2206 treatment. Combination treatment was also found tobe significantly more effective than either treatment alone at reducingcolony formation in cancer cells (FIG. 19) and in inducing apoptosis inthese cells (FIG. 20). The ability of TUSC2 therapy to sensitize cellsto AKT protein kinase therapy was specifically quantified in Table 13,which shows that TUSC2 treatment reduced the effective IC₅₀ of MK2206 atleast 5-fold and, it some cases, by as much as 16-fold. Furthermore thecombined effectiveness of the therapies was confirmed in vivo using amurine tumor explants model. As shown in FIG. 24, the combinedadministration of TUSC2 nanoparticles and MK2206 was far more effectivethan each therapy in isolation at preventing tumor growth and tumorgrowth in co-treated animals was infect very minimal. Thus, the TUSC2therapies described here can be combined with protein kinase inhibitortherapies to further increase the effectiveness of these inhibitors andeven to reverse resistance to such agents in cancers.

I. Assessing Apoptosis

As detailed above, methods for determining the proportion of apoptoticcancer cells in a subject can be useful predicting a response to TUSC2therapy. Assessment of apoptosis may be performed on a sample of cancercells from the subject or in vivo assessment may be performed (e.g., byimaging). For example, methods for in vivo assessment of apoptosis wererecently review by Blankenberg 2008 and Zhao 2009 (both of which areincorporated herein by reference). A wide range of methods may beemployed to identify apoptotic cells, ranging from simple lightmicroscopy to molecular assays that detect changes in cellular membraneintegrity, changes in cellular gene expression, activation proteases andDNA fragmentation.

In certain aspects, the proportion (i.e., percentage) of cells in asample that are apoptotic. However, it will be recognized that not allmethods for determining apoptosis provide an assessment on acell-by-cell basis. Thus, in certain aspects, a level of apoptosis isdetermined for a sample, wherein the level correlates with a particularportion of apoptotic cells (e.g., at least about 10% apoptotic cells).For example, level of apoptosis may be determine for the cancer cells ofpatient (e.g., the intensity of in vivo Annexin V staining) and thelevel correlated to a percentage of apoptotic cells to determine withthe subject will response favorably to a TUSC2 therapy.

a. DNA fragmentation

During apoptosis nuclear DNA is fragmented and these changes can bedetected to assess apoptosis in a sample. Fragmented DNA may bedetected, for example, by light microscopy, which can revealcondensation and margination of chromatin. Fragmentation of DNA can alsobe directly assessed using a separative method, e.g., chromatography orelectrophoresis, to size fractionate the sample. For example, DNAfragmentation, characteristic of apoptosis, will be visualized as“ladders” containing a wide range of fragments. Use of such methods,however, may not provide the best quantitative assessment of apoptosis.

Apoptotic cells can also be detected by end labeling of fragmented DNA.For instance, apoptosis can be assayed using terminaldeoxytransferase-mediated (TdT) dUTP biotin nick end-labeling (TUNEL;Gavriel et al., J. Cell Biol. 119:493 (1992); Gorczyca et al., Int. J.Oncol. 1:639 (1992). TUNEL labeling is effected by incorporation oflabeled nucleotides into the 3′ hydroxyl termini of the DNA breakscharacteristic of apoptosis using the enzyme terminal transferase. Theincorporated nucleotide may be labeled by a wide variety of techniques.A typical approach is to incorporate a ligand such as fluorescein,biotin or digoxigenin into the nucleotide. If the ligand itself is notcapable of yielding a signal, typically fluorescence, it can be reactedwith a second moiety such as an appropriate antibody or other receptorwhich does carry a signal generator after incorporation of thenucleotide into the DNA terminal. Typical of such an approach is the useof a digoxigenin carrying nucleotide with the later reaction with ananti-digoxigenin antibody carrying Rhodamine, or a bromolated nucleotidewith the later reaction with an appropriate antibody carryingfluorescein.

A similar labeling method is know as in situ end-labeling (INSEL). ForINSEL, labeling is effected in a similar manner to TUNEL labeling exceptthat the labeled nucleotide is incorporated using the enzyme DNApolymerase I or its Klenow fragment. It is general this method may besomewhat less sensitive and specific than TUNEL labeling.

Both TUNEL and INSEL labeling require that certain steps be taken inorder to have the labeled nucleotides access the nuclear DNA of thecells being analyzed. These steps are well known and included in theinstructions accompanying the commercial kits. In general they involverendering the cell walls of the cells being analyzed permeable to thelabeled nucleotide and incorporating enzyme and removing any proteinmasking by appropriate protein digestion such as with pepsin.

b. Lose of Membrane Integrity

During apoptosis membrane integrity of the plasma membrane andmitochondrial membrane is altered and these alterations can be detectedto identify apoptotic cells. For example, light microscopy may be usedto determine the presence of one or more morphological characteristicsof apoptosis such as condensed or rounded morphology, shrinking andblebbing of the cytoplasm. Likewise, certain membrane constituents canbecome exposed to the exterior of the cell and detected as an indicatorof apoptosis.

Detection of phosphatidylserine on the exterior of cells can beindicative of apoptosis. For example, commercial kits are available forthe detection of phosphatidylserine via Annexin V binding (see, e.g.,the FITC Annexin V Apoptosis Kit available from BD Pharmingen™). LabeledAnnexin V, such as radiolabeled Annexin V, may also be used for in vivoimaging of cancer cells to assess apoptosis (see, e.g., Blankenberg2008).

Permeablization of the mitochondrial membrane is also an indicator ofapoptosis. Once, mitochondrial membrane integrity is lost certainproteins are released to the cytoplasm and detection of such proteinsmay be use to assess apoptosis. For example, detection of cytochrome c(Cyt c) release is a commonly used apoptotic indicator.

c. Caspase Activation

Members of the caspase family of proteins are major effectors ofcellular apoptosis. Caspases are cysteine proteases that exist withinthe cell as inactive pro-forms or so-called “zymogens.” The zymogens arecleaved to form active enzymes following the induction of apoptosiseither via the death receptor-mediated pathway or the mitochondrialpathway of apoptosis. Depending upon the apoptotic pathway, differentcaspases initiate the apoptotic process, with Caspase-8 and -10initiating the death receptor pathway, and Caspase-9 initiating themitochondrial pathway. Active initiator caspases then activate (i.e.,cleave) effector caspases, for example, Caspase-3, -6, and -7, to induceapoptosis. These effector caspases cleave key cellular proteins thatlead to the typical morphological changes observed in cells undergoingapoptosis. Thus, in certain aspects, apoptosis can be detected bydirectly detecting caspase activity (e.g., by use of fluorescentlylabeled peptides with a caspase cleavage site) or indirectly detectingthe activated enzymes by detecting a cleaved target polypeptide.

One protein often used to indirectly detect caspase activity ispoly(ADP-ribose) polymerase (PARP-1). PARP-1 is a DNA-binding proteinthat is specifically cleaved during apoptosis. Active PARP-1 catalyzesthe addition of poly(ADP-ribose) chains to some nuclear proteins and isthought to play a critical role in DNA damage repair. PARP-1 is rapidlyactivated during cellular stresses, such as heat shock, ionizingradiation, exposure to carcinogens, and treatment with chemotherapyagents. During apoptosis, activated (i.e., cleaved) caspase-3 in turncleaves PARP-1. Thus, the presence and indeed the level of cleavedPARP-1 can be used to assess apoptosis in a sample.

d. Changes in Gene Expression

A variety of additional changes in cellular gene expression occur duringapoptosis and can be detected as indicators of apoptosis. For example,the expression of pro-apoptotic proteins such as Bid, Bim, Bik, Bmf,Bad, Hrk, BNIP3, Bax, Bak, and Bok may be used as an apoptotic marker.

II. Nucleic Acid and Polypeptide Complexes

In certain aspects, concerns compositions and methods for delivering anucleic acid or a polypeptide to a cell. In particular, provided hereinare nanoparticle-nucleic acid or nanoparticle-polypeptide complexes andmethods of administering such complexes to a subject. The complexescomprise a TUSC2 polypeptide and/or nucleic acid in association with ananoparticle. As used herein, “association” means a physicalassociation, a chemical association or both. For example, an associationcan involve a covalent bond, a hydrophobic interaction, encapsulation,surface adsorption, or the like.

Polypeptides and nucleic acids typically have difficulty crossingcellular membranes. Both types of molecules include charged residues,which hinder membrane binding and membrane transport into cells. Thepresent embodiments overcome this difficulty by, providing nanoparticlecomplexes that facilitate cellular uptake.

In accordance with the present embodiments, a polypeptide and/or nucleicacid may be associated with a nanoparticle to form nanoparticle complex.In some embodiments, the nanoparticle is a liposomes or otherlipid-based nanoparticle such as a lipid-based vesicle (e.g., aDOTAP:cholesterol vesicle). As used in cancer therapy, liposomes takeadvantage of the increased fenestrations in the cancer neovasculature toenhance liposome concentration at tumor sites.

In other embodiments, the nanoparticle is a non-lipid nanoparticle, suchas an iron-oxide based superparamagnetic nanoparticles.Superparamagnetic nanoparticles ranging in diameter from about 10 to 100nm are small enough to avoid sequestering by the spleen, but largeenough to avoid clearance by the liver. Particles this size canpenetrate very small capillaries and can be effectively distributed inbody tissues. Superparamagnetic nanoparticles complexes can be used asMM contrast agents to identify and follow those cells that take up thetherapeutic complexes. In certain embodiments, the nanoparticle is asemiconductor nanocrystal or a semiconductor quantum dot, both of whichcan be used in optical imaging. In further embodiments, the nanoparticlecan be a nanoshell, which comprises a gold layer over a core of silica.One advantage of nanoshells is that a polypeptide or nucleic acid can beconjugated to the gold layer using standard chemistry. In otherembodiments, the nanoparticle can be a fullerene or a nanotube (Gupta etal., 2005).

In accordance with the present embodiments, nanoparticle complexes canbe targeted to specific tissues and cells. This can be accomplished byconjugating a cell targeting moiety to the nanoparticle. The targetingmoiety can be, but is not limited to, a protein, peptide, lipid,steroid, sugar, carbohydrate or synthetic compound. Cell targetingmoieties such as ligands recognize and bind to their cognate receptorson the surface of cells. Similarly, antibody can act as cell targetingmoieties by recognizing their cognate antigens on the cell surface. Incertain embodiments, targeted nanoparticle complexes provided herein canenhance the specificity of disease treatment and increase the amount oftherapeutic agent entering a targeted cell.

a. Nanoparticles

As used herein, the term “nanoparticle” refers to any material havingdimensions in the 1-1,000 nm range. In some embodiments, nanoparticleshave dimensions in the 50-500 nm range. Nanoparticles used in thepresent embodiments include such nanoscale materials as a lipid-basednanoparticle, a superparamagnetic nanoparticle, a nanoshell, asemiconductor nanocrystal, a quantum dot, a polymer-based nanoparticle,a silicon-based nanoparticle, a silica-based nanoparticle, a metal-basednanoparticle, a fullerene and a nanotube (Ferrari, 2005). Theconjugation of polypeptide or nucleic acids to nanoparticles providesstructures with potential application for targeted delivery, controlledrelease, enhanced cellular uptake and intracellular trafficking, andmolecular imaging of therapeutic peptides in vitro and in vivo (West,2004; Stayton et al., 2000; Ballou et al., 2004; Frangioni, 2003;Dubertret et al., 2002; Michalet et al., 2005; Dwarakanath et al., 2004.

1. Lipid-Based Nanoparticles

Lipid-based nanoparticles include liposomes, lipid preparations andlipid-based vesicles (e.g., DOTAP:cholesterol vesicles). Lipid-basednanoparticles may be positively charged, negatively charged or neutral.In certain embodiments, the lipid-based nanoparticle is neutrallycharged (e.g., a DOPC liposome).

A “liposome” is a generic term encompassing a variety of single andmultilamellar lipid vehicles formed by the generation of enclosed lipidbilayers or aggregates. Liposomes may be characterized as havingvesicular structures with a bilayer membrane, generally comprising aphospholipid, and an inner medium that generally comprises an aqueouscomposition. Liposomes provided herein include unilamellar liposomes,multilamellar liposomes and multivesicular liposomes. Liposomes providedherein may be positively charged, negatively charged or neutrallycharged. In certain embodiments, the liposomes are neutral in charge.

A multilamellar liposome has multiple lipid layers separated by aqueousmedium. They form spontaneously when lipids comprising phospholipids aresuspended in an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Lipophilic molecules or molecules with lipophilicregions may also dissolve in or associate with the lipid bilayer.

In specific aspects, a polypeptide or nucleic acids may be, for example,encapsulated in the aqueous interior of a liposome, interspersed withinthe lipid bilayer of a liposome, attached to a liposome via a linkingmolecule that is associated with both the liposome and thepolypeptide/nucleic acid, entrapped in a liposome, complexed with aliposome, or the like.

A liposome used according to the present embodiments can be made bydifferent methods, as would be known to one of ordinary skill in theart. For example, a phospholipid (Avanti Polar Lipids, Alabaster, Ala.),such as for example the neutral phospholipid dioleoylphosphatidylcholine(DOPC), is dissolved in tert-butanol. The lipid(s) is then mixed with apolypeptide, nucleic acid, and/or other component(s). Tween 20 is addedto the lipid mixture such that Tween 20 is about 5% of the composition'sweight. Excess tert-butanol is added to this mixture such that thevolume of tert-butanol is at least 95%. The mixture is vortexed, frozenin a dry ice/acetone bath and lyophilized overnight. The lyophilizedpreparation is stored at −20° C. and can be used up to three months.When required the lyophilized liposomes are reconstituted in 0.9%saline.

Alternatively, a liposome can be prepared by mixing lipids in a solventin a container, e.g., a glass, pear-shaped flask. The container shouldhave a volume ten-times greater than the volume of the expectedsuspension of liposomes. Using a rotary evaporator, the solvent isremoved at approximately 40° C. under negative pressure. The solventnormally is removed within about 5 min. to 2 hours, depending on thedesired volume of the liposomes. The composition can be dried further ina desiccator under vacuum. The dried lipids generally are discardedafter about 1 week because of a tendency to deteriorate with time.

Dried lipids can be hydrated at approximately 25-50 mM phospholipid insterile, pyrogen-free water by shaking until all the lipid film isresuspended. The aqueous liposomes can be then separated into aliquots,each placed in a vial, lyophilized and sealed under vacuum.

The dried lipids or lyophilized liposomes prepared as described abovemay be dehydrated and reconstituted in a solution of a protein orpeptide and diluted to an appropriate concentration with an suitablesolvent, e.g., DPBS. The mixture is then vigorously shaken in a vortexmixer. Unencapsulated additional materials, such as agents including butnot limited to hormones, drugs, nucleic acid constructs and the like,are removed by centrifugation at 29,000×g and the liposomal pelletswashed. The washed liposomes are resuspended at an appropriate totalphospholipid concentration, e.g., about 50-200 mM. The amount ofadditional material or active agent encapsulated can be determined inaccordance with standard methods. After determination of the amount ofadditional material or active agent encapsulated in the liposomepreparation, the liposomes may be diluted to appropriate concentrationsand stored at 4° C. until use. A pharmaceutical composition comprisingthe liposomes will usually include a sterile, pharmaceuticallyacceptable carrier or diluent, such as water or saline solution.

In other alternative methods, liposomes can be prepared in accordancewith other known laboratory procedures (e.g., see Bangham et al., 1965;Gregoriadis, 1979; Deamer and Uster, 1983; Szoka and Papahadjopoulos,1978, each incorporated herein by reference in relevant part).Additional liposomes which may be useful with the present embodimentsinclude cationic liposomes, for example, as described in WO02/100435A1,U.S. Pat. No. 5,962,016, U.S. Application 2004/0208921, WO03/015757A1,WO04029213A2, U.S. Pat. No. 5,030,453, and U.S. Pat. No. 6,680,068, allof which are hereby incorporated by reference in their entirety withoutdisclaimer. A process of making liposomes is also described inWO04/002453A1. Neutral lipids can be incorporated into cationicliposomes (e.g., Farhood et al., 1995). Various neutral liposomes whichmay be used in certain embodiments are disclosed in U.S. Pat. No.5,855,911, which is incorporated herein by reference. These methodsdiffer in their respective abilities to entrap aqueous material andtheir respective aqueous space-to-lipid ratios.

The size of a liposome varies depending on the method of synthesis.Liposomes in the present embodiments can be a variety of sizes. Incertain embodiments, the liposomes are small, e.g., less than about 100nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, or less thanabout 50 nm in external diameter. For example, in general, prior to theincorporation of nucleic acid, a DOTAP:cholesterol liposome for useaccording to the present embodiments comprises a size of about 50 to 500nm. Such liposome formulations may also be defined by particle charge(zeta potential) and/or optical density (OD). For instance, aDOTAP:cholesterol liposome formulation will typically comprise an OD₄₀₀of less than 0.45 prior to nucleic acid incorporation. Likewise, theoverall charge of such particles in solution can be defined by a zetapotential of about 50-80 mV.

In preparing such liposomes, any protocol described herein, or as wouldbe known to one of ordinary skill in the art may be used. Additionalnon-limiting examples of preparing liposomes are described in U.S. Pat.Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505,and 4,921,706; International Applications PCT/US85/01161 andPCT/US89/05040; U.K. Patent Application GB 2193095 A; Mayer et al.,1986; Hope et al., 1985; Mayhew et al. 1987; Mayhew et al., 1984; Chenget al., 1987; and Liposome Technology, 1984, each incorporated herein byreference).

In certain embodiments, the lipid based nanoparticle is a neutralliposome (e.g., a DOPC liposome). “Neutral liposomes” or “non-chargedliposomes”, as used herein, are defined as liposomes having one or morelipid components that yield an essentially-neutral, net charge(substantially non-charged). By “essentially neutral” or “essentiallynon-charged”, it is meant that few, if any, lipid components within agiven population (e.g., a population of liposomes) include a charge thatis not canceled by an opposite charge of another component (i.e., fewerthan 10% of components include a non-canceled charge, more preferablyfewer than 5%, and most preferably fewer than 1%). In certainembodiments, neutral liposomes may include mostly lipids and/orphospholipids that are themselves neutral under physiological conditions(i.e., at about pH 7).

Liposomes and/or lipid-based nanoparticles of the present embodimentsmay comprise a phospholipid. In certain embodiments, a single kind ofphospholipid may be used in the creation of liposomes (e.g., a neutralphospholipid, such as DOPC, may be used to generate neutral liposomes).In other embodiments, more than one kind of phospholipid may be used tocreate liposomes.

Phospholipids include, for example, phosphatidylcholines,phosphatidylglycerols, and phosphatidylethanolamines; becausephosphatidylethanolamines and phosphatidyl cholines are non-chargedunder physiological conditions (i.e., at about pH 7), these compoundsmay be particularly useful for generating neutral liposomes. In certainembodiments, the phospholipid DOPC is used to produce non-chargedliposomes. In certain embodiments, a lipid that is not a phospholipid(e.g., a cholesterol) may be used

Phospholipids include glycerophospholipids and certain sphingolipids.Phospholipids include, but are not limited to,dioleoylphosphatidylycholine (“DOPC”), egg phosphatidylcholine (“EPC”),dilauryloylphosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine(“DMPC”), dipalmitoylphosphatidylcholine (“DPPC”),distearoylphosphatidylcholine (“DSPC”), 1-myristoyl-2-palmitoylphosphatidylcholine (“WPC”), 1-palmitoyl-2-myri stoylphosphatidylcholine (“PMPC”), 1-palmitoyl-2-stearoyl phosphatidylcholine(“PSPC”), 1-stearoyl-2-palmitoyl phosphatidylcholine (“SPPC”),dilauryloylphosphatidylglycerol (“DLPG”),dimyristoylphosphatidylglycerol (“DWG”), dipalmitoylphosphatidylglycerol(“DPPG”), distearoylphosphatidylglycerol (“DSPG”), di stearoylsphingomyelin (“DSSP”), distearoylphophatidylethanolamine (“DSPE”),dioleoylphosphatidylglycerol (“DOPG”), dimyristoyl phosphatidic acid(“DMPA”), dipalmitoyl phosphatidic acid (“DPPA”), dimyristoylphosphatidylethanolamine (“DMPE”), dipalmitoyl phosphatidylethanolamine(“DPPE”), dimyristoyl phosphatidylserine (“DMPS”), dipalmitoylphosphatidylserine (“DPPS”), brain phosphatidylserine (“BPS”), brainsphingomyelin (“BSP”), dipalmitoyl sphingomyelin (“DPSP”), dimyristylphosphatidylcholine (“DMPC”), 1,2-distearoyl-sn-glycero-3-phosphocholine(“DAPC”), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (“DBPC”),1,2-dieicosenoyl-sn-glycero-3-phosphocholine (“DEPC”),dioleoylphosphatidylethanolamine (“DOPE”), palmitoyloeoylphosphatidylcholine (“POPC”), palmitoyloeoyl phosphatidylethanolamine(“POPE”), lysophosphatidylcholine, lysophosphatidylethanolamine, anddilinoleoylphosphatidylcholine.

Phospholipids may be from natural or synthetic sources. However,phospholipids from natural sources, such as egg or soybeanphosphatidylcholine, brain phosphatidic acid, brain or plantphosphatidylinositol, heart cardiolipin and plant or bacterialphosphatidylethanolamine are not used, in certain embodiments, as theprimary phosphatide (i.e., constituting 50% or more of the totalphosphatide composition) because this may result in instability andleakiness of the resulting liposomes.

2. DOTAP:Cholesterol Nanoparticle

In certain embodiments, the lipid-based vesicle is a DOTAP:cholesterolnanoparticle. DOTAP:cholesterol nanoparticles are prepared by mixing thecationic lipid DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)-propane)with cholesterol. Vesicles prepared with DNA can form a structure(called a “sandwich”) where the DNA appears to be condensed between twolipid bilayers (U.S. Pat. Nos. 6,770,291 and 6,413,544).

A DOTAP:cholesterol-nucleic acid complex can be prepared as in thefollowing non-limiting example. The DOTAP:cholesterol (DC) nanoparticles(sized 50 to 500 nm) are synthesized as described previously (U.S. Pat.Nos. 6,770,291 and 6,413,544; Templeton, 1997). Briefly, 420 mg of DOTAPand 208 mg of cholesterol are measure and mixed together with 30 ml ofchloroform. Mixture is then allowed to dry on a rotary evaporator for 30minutes and freeze dry for 15 minutes. The dried mixture isreconstituted in 30 ml of D5W by swirling at 50° C. for 45 minutes and37° C. for 10 minutes. The mixture is ten subjected to low frequencysonication for five minutes to form liposomes. DOTAP:cholesterolliposome are then heated to 50° C. and sequentially filtered through1.0, 0.45, 0.2 and 0.1 μm sterile Whatman filters. The synthesizednanoparticles are stored at 4° C. and used for preparing nanoparticlecomplexes. The formulated DOTAP:cholesterol liposome should be evenlydispersed with a particle size of 50-250 nm, an OD₄₀₀ of less than 0.45and zeta potential of 50-80 mV. Residual CHCl₃ levels should be lessthan 60 ppm.

To prepare DOTAP:cholesterol-nucleic acid nanoparticles, 240 μl ofliposomes (see above) are diluted in 360 μl D5W at room temperature. DNA(˜5 mg/ml) is added to the mixture to a total volume of 600 μl. Themixture is moved up and down in a pipet to mix. Once settled the mixtureshould have a an OD₄₀₀ of between 0.65 and 0.95, a particle size of200-500 nm and be confirmed gram stain negative. The liposome complexesare stored at between 3° C. and 28° C. and agitated as little aspossible.

b. Targeting of Nanoparticles

Targeted delivery is achieved by the addition of ligands withoutcompromising the ability of nanoparticles to deliver their payloads. Itis contemplated that this will enable delivery to specific cells,tissues and organs. The targeting specificity of the ligand-baseddelivery systems are based on the distribution of the ligand receptorson different cell types. The targeting ligand may either benon-covalently or covalently associated with a nanoparticle, and can beconjugated to the nanoparticles by a variety of methods as discussedherein.

Examples of proteins or peptides that can be used to targetnanoparticles include transferin, lactoferrin, TGF-α, nerve growthfactor, albumin, HIV Tat peptide, RGD peptide, and insulin, as well asothers (Gupta et al., 2005; Ferrari, 2005).

III. TUSC2 Expression Vectors

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (see, for example, Maniatis et al., 1989 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

In certain embodiments, provided herein is the use of nucleic acidsTUSC2 coding sequence. For example, such vector can be used forrecombinant production of a TUSC2 polypeptide and/or for the expressionof TUSC2 in vivo in a subject. The sequences may be modified, given theability of several different codons to encode a single amino acid, whilestill encoding for the same protein or polypeptide. Optimization ofcodon selection can also be undertaken in light of the particularorganism used for recombinant expression or may be optimized for maximalexpression in human cell (e.g., a cancer cell). Vector for use inaccordance with the present embodiments additionally comprise elementsthat control gene expression and/or aid in vector production andpurification.

a. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30-110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous” or “homologous.” Similarly, an enhancer maybe one naturally associated with a nucleic acid sequence, located eitherdownstream or upstream of that sequence. Alternatively, certainadvantages will be gained by positioning the coding nucleic acid segmentunder the control of a recombinant, exogenous or heterologous promoter,which refers to a promoter that is not normally associated with anucleic acid sequence in its natural environment. A recombinant orheterologous enhancer refers also to an enhancer not normally associatedwith a nucleic acid sequence in its natural environment. Such promotersor enhancers may include viral promoter and enhancers such as the CMVpromoter.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell, tissue, organ, or organism chosen for expression. Thoseof skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al. 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

Additionally any promoter/enhancer combination (as per, for example, theEukaryotic Promoter Data Base EPDB, www.epd.isb-sib.ch/) could also beused to drive expression. Use of a T3, T7 or SP6 cytoplasmic expressionsystem is another possible embodiment. Eukaryotic cells can supportcytoplasmic transcription from certain bacterial promoters if theappropriate bacterial polymerase is provided, either as part of thedelivery complex or as an additional genetic expression construct.

b. Translation Initiation Signals

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

c. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector (see, for example, Carbonelli et al., 1999, Levensonet al., 1998, and Cocea, 1997, incorporated herein by reference).“Restriction enzyme digestion” refers to catalytic cleavage of a nucleicacid molecule with an enzyme that functions only at specific locationsin a nucleic acid molecule. Many of these restriction enzymes arecommercially available. Use of such enzymes is widely understood bythose of skill in the art. Frequently, a vector is linearized orfragmented using a restriction enzyme that cuts within the MCS to enableexogenous sequences to be ligated to the vector. “Ligation” refers tothe process of forming phosphodiester bonds between two nucleic acidfragments, which may or may not be contiguous with each other.Techniques involving restriction enzymes and ligation reactions are wellknown to those of skill in the art of recombinant technology.

d. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, Chandler et al., 1997, herein incorporated by reference).Inclusion of such splice sites also can enhance expression by avertingnon-sense mediated decay of resulting RNA transcripts.

e. Termination Signals

The vectors or constructs of the present embodiments will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

Terminators contemplated for use in the present embodiments include anyknown terminator of transcription described herein or known to one ofordinary skill in the art, including but not limited to, for example,the termination sequences of genes, such as for example the bovinegrowth hormone terminator or viral termination sequences, such as forexample the SV40 terminator. In certain embodiments, the terminationsignal may be a lack of transcribable or translatable sequence, such asdue to a sequence truncation.

f. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the present embodiments, andany such sequence may be employed. Preferred embodiments include theSV40 polyadenylation signal or the bovine growth hormone polyadenylationsignal, convenient and known to function well in various target cells.Polyadenylation may increase the stability of the transcript or mayfacilitate cytoplasmic transport.

g. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

h. Selectable and Screenable Markers

In certain embodiments, cells containing a nucleic acid constructprovided herein may be identified in vitro or in vivo by including amarker in the expression vector. Such markers would confer anidentifiable change to the cell permitting easy identification of cellscontaining the expression vector. Generally, a selectable marker is onethat confers a property that allows for selection. A positive selectablemarker is one in which the presence of the marker allows for itsselection, while a negative selectable marker is one in which itspresence prevents its selection. An example of a positive selectablemarker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

i. Plasmid Vectors

In certain embodiments, a plasmid vector is contemplated for use totransform a host cell. In general, plasmid vectors containing repliconand control sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries a replication site, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. In anon-limiting example, E. coli is often transformed using derivatives ofpBR322, a plasmid derived from an E. coli species. pBR322 contains genesfor ampicillin and tetracycline resistance and thus provides easy meansfor identifying transformed cells. The pBR plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, forexample, promoters which can be used by the microbial organism forexpression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEM™-11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as, for example,E. coli LE392.

Further useful plasmid vectors include pIN vectors (Inouye et al.,1985); and pGEX vectors, for use in generating glutathione S-transferase(GST) soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with β-galactosidase,ubiquitin, and the like.

Bacterial host cells, for example, E. coli, comprising the expressionvector, are grown in any of a number of suitable media, for example, LB.The expression of the recombinant protein in certain vectors may beinduced, as would be understood by those of skill in the art, bycontacting a host cell with an agent specific for certain promoters,e.g., by adding IPTG to the media or by switching incubation to a highertemperature. After culturing the bacteria for a further period,generally of 2-24 hr, the cells are collected by centrifugation andwashed to remove residual media.

j. Viral Vectors

The ability of certain viruses to infect cells or enter cells viareceptor-mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). Viruses may thus be utilized that encode and expressTUSC2. Non-limiting examples of virus vectors that may be used todeliver a TUSC2 nucleic acid are described below.

Adenoviral Vectors.

A particular method for delivery of the nucleic acid involves the use ofan adenovirus expression vector. Although adenovirus vectors are knownto have a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a tissue orcell-specific construct that has been cloned therein. Knowledge of thegenetic organization or adenovirus, a 36 kb, linear, double-stranded DNAvirus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus and Horwitz, 1992).

AAV Vectors.

The nucleic acid may be introduced into the cell using adenovirusassisted transfection. Increased transfection efficiencies have beenreported in cell systems using adenovirus coupled systems (Kelleher andVos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated virus(AAV) has a high frequency of integration and it can infect non-dividingcells, thus making it useful for delivery of genes into mammalian cells,for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has abroad host range for infectivity (Tratschin et al., 1984; Laughlin etal., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Detailsconcerning the generation and use of rAAV vectors are described in U.S.Pat. Nos. 5,139,941 and 4,797,368, each incorporated herein byreference.

Retroviral Vectors.

Retroviruses have the ability to integrate their genes into the hostgenome, transferring a large amount of foreign genetic material,infecting a broad spectrum of species and cell types and of beingpackaged in special cell-lines (Miller, 1992). In order to construct aretroviral vector, a nucleic acid (e.g., one encoding a protein ofinterest) is inserted into the viral genome in the place of certainviral sequences to produce a virus that is replication-defective. Inorder to produce virions, a packaging cell line containing the gag, pol,and env genes but without the LTR and packaging components isconstructed (Mann et al., 1983). When a recombinant plasmid containing acDNA, together with the retroviral LTR and packaging sequences isintroduced into a special cell line (e.g., by calcium phosphateprecipitation for example), the packaging sequence allows the RNAtranscript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubinstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomeret al., 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 andthe Simian Immunodeficiency Virus: SIV. Lentiviral vectors have beengenerated by multiply attenuating the HIV virulence genes, for example,the genes env, vif, vpr, vpu and nef are deleted making the vectorbiologically safe.

Other Viral Vectors.

Other viral vectors may be employed as vaccine constructs in the presentembodiments. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988),sindbis virus, cytomegalovirus and herpes simplex virus may be employed.They offer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

Modified Viruses.

A nucleic acid to be delivered may be housed within an infective virusthat has been engineered to express a specific binding ligand. The virusparticle will thus bind specifically to the cognate receptors of thetarget cell and deliver the contents to the cell. A novel approachdesigned to allow specific targeting of retrovirus vectors was developedbased on the chemical modification of a retrovirus by the chemicaladdition of lactose residues to the viral envelope. This modificationcan permit the specific infection of hepatocytes via sialoglycoproteinreceptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et al., 1989).

IV. Pharmaceutical Formulations

Pharmaceutical compositions provided herein comprise an effective amountof one or more TUSC2 therapeutic and, optionally, an additional agentdissolved or dispersed in a pharmaceutically acceptable carrier. Thephrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of apharmaceutical composition that contains at least TUSC2 nucleic acid,peptide or a nanoparticle complex or additional active ingredient willbe known to those of skill in the art in light of the presentdisclosure, as exemplified by Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference.Moreover, for animal (e.g., human) administration, it will be understoodthat preparations should meet sterility, pyrogenicity, general safetyand purity standards as required by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

In certain embodiments, the pharmaceutical composition may comprisedifferent types of carriers depending on whether it is to beadministered in solid, liquid or aerosol form, and whether it need to besterile for such routes of administration as injection. In certainembodiments, pharmaceutical compositions provided herein can beadministered intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intranasally,intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally,inhalation (e.g. aerosol inhalation), injection, infusion, continuousinfusion, localized perfusion bathing target cells directly, via acatheter, via a lavage, in cremes, in lipid compositions (e.g.,liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference).

In certain embodiments, the pharmaceutical composition is administeredintraperitoneally. In further embodiments, the pharmaceuticalcomposition is administered intraperitoneally to treat a cancer (e.g., acancerous tumor). For example, the pharmaceutical composition may beadministered intraperitoneally to treat gastrointestinal cancer. Incertain embodiments it may be desirable to administer the pharmaceuticalcomposition into or near a tumor.

In certain preferred embodiments, the pharmaceutical composition isadministered orally to treat a cancer (e.g., a gastrointestinal cancer).

In certain embodiments, the actual dosage amount of a compositionadministered to a patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 15 microgram/kg/body weight, about 20 microgram/kg/bodyweight, about 25 microgram/kg/body weight, about 30 microgram/kg/bodyweight, about 35 microgram/kg/body weight, about 0.04 milligram/kg/bodyweight, about 0.05 milligram/kg/body weight, about 0.06milligram/kg/body weight, about 0.07 milligram/kg/body weight, about0.08 milligram/kg/body weight, about 0.09 milligram/kg/body weight,about 0.1 milligram/kg/body weight, about 0.2 milligram/kg/body weight,to about 0.5 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 0.01 mg/kg/body weight toabout 0.1 mg/kg/body weight, about 0.04 microgram/kg/body weight toabout 0.08 milligram/kg/body weight, etc., can be administered, based onthe numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The one or more peptides, nanoparticle complexes or additional agent maybe formulated into a composition in a free base, neutral or salt form.Pharmaceutically acceptable salts, include the acid addition salts,e.g., those formed with the free amino groups of a proteinaceouscomposition, or which are formed with inorganic acids such as forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols or inhalants in the present embodiments. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in preferred embodiments the aqueous nasal solutions usually areisotonic or slightly buffered to maintain a pH of about 5.5 to about6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments the one or more polypeptide, nucleic acid ornanoparticle complexes are prepared for administration by such routes asoral ingestion. In these embodiments, the solid composition maycomprise, for example, solutions, suspensions, emulsions, tablets,pills, capsules (e.g., hard or soft shelled gelatin capsules), sustainedrelease formulations, buccal compositions, troches, elixirs,suspensions, syrups, wafers, or combinations thereof. Oral compositionsmay be incorporated directly with the food of the diet. Preferredcarriers for oral administration comprise inert diluents, assimilableedible carriers or combinations thereof. In other aspects, the oralcomposition may be prepared as a syrup or elixir. A syrup or elixir, andmay comprise, for example, at least one active agent, a sweeteningagent, a preservative, a flavoring agent, a dye, a preservative, orcombinations thereof

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

V. Combination Therapies

In order to increase the effectiveness of a nucleic acid, polypeptide ornanoparticle complex of the present embodiments, it may be desirable tocombine these compositions with other agents effective in the treatmentof the disease of interest.

As a non-limiting example, the treatment of cancer may be implementedwith TUSC2 therapeutic of the present embodiments along with otheranti-cancer agents. An “anti-cancer” agent is capable of negativelyaffecting cancer in a subject, for example, by killing cancer cells,inducing apoptosis in cancer cells, reducing the growth rate of cancercells, reducing the incidence or number of metastases, reducing tumorsize, inhibiting tumor growth, reducing the blood supply to a tumor orcancer cells, promoting an immune response against cancer cells or atumor, preventing or inhibiting the progression of cancer, or increasingthe lifespan of a subject with cancer. More generally, these othercompositions would be provided in a combined amount effective to kill orinhibit proliferation of the cell. This process may involve contactingthe cells with the anti-cancer peptide or nanoparticle complex and theagent(s) or multiple factor(s) at the same time. This may be achieved bycontacting the cell with a single composition or pharmacologicalformulation that includes both agents, or by contacting the cell withtwo distinct compositions or formulations, at the same time, wherein onecomposition includes the anti-cancer peptide or nanoparticle complex andthe other includes the second agent(s). In particular embodiments, ananti-cancer peptide can be one agent, and an anti-cancer nanoparticlecomplex can be the other agent.

Treatment with the anti-cancer peptide or nanoparticle-complex mayprecede or follow the other agent treatment by intervals ranging fromminutes to weeks. In embodiments where the other agent and theanti-cancer peptide or nanoparticle complex are applied separately tothe cell, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the agentand the anti-cancer peptide or nanoparticle complex would still be ableto exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one may contact the cell with bothmodalities within about 12-24 hours of each other and, more preferably,within about 6-12 hours of each other. In some situations, it may bedesirable to extend the time period for treatment significantly whereseveral days (e.g., 2, 3, 4, 5, 6 or 7 days) to several weeks (e.g., 1,2, 3, 4, 5, 6, 7 or 8 weeks) lapse between the respectiveadministrations.

Likewise, in certain aspects a TUSC2 therapy is administered inconjunction with an anti-inflammatory agent. For example, a TUSC2therapy may precede or follow the anti-inflammatory agent treatment byintervals ranging from minutes to weeks. In certain aspects, theanti-inflammatory agent is administered immediately before the TUSC2therapy and immediately after the TUSC2 therapy. For example, theanti-inflammatory agent may be given less than a day before and lessthan a day after the therapy. In still further aspects more than oneanti-inflammatory is administered, such administration of aantihistamine (e.g., diphenhydramine) and a corticosteroid (e.g.,dexamethasone).

Various combinations may be employed, where the TUSC2 therapy is “A” andthe secondary agent, such as radiotherapy, chemotherapy oranti-inflammatory agent, is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/ABA/A/B/B A/B/AB A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

In certain embodiments, administration of the TUSC2 therapy of thepresent embodiments to a patient will follow general protocols for theadministration of chemotherapeutics, taking into account the toxicity,if any, of the vector. It is expected that the treatment cycles would berepeated as necessary. It also is contemplated that various standardtherapies, as well as surgical intervention, may be applied incombination with the described hyperproliferative cell therapy.

a. Chemotherapy

Cancer therapies also include a variety of combination therapies. Insome aspects a TUSC2 therapeutic of the embodiments is administered (orformulated) in conjunction with a chemotherapeutic agent. For example,in some aspects the chemotherapeutic agent is a protein kinase inhibitorsuch as a EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src,GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor or BRAFinhibitors. Nonlimiting examples of protein kinase inhibitors includeAfatinib, Axitinib, Bevacizumab, Bosutinib, Cetuximab, Crizotinib,Dasatinib, Erlotinib, Fostamatinib, Gefitinib, Imatinib, Lapatinib,Lenvatinib, Mubritinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib,Ranibizumab, Ruxolitinib, Saracatinib, Sorafenib, Sunitinib,Trastuzumab, Vandetanib, AP23451, Vemurafenib, MK-2206, GSK690693,A-443654, VQD-002, Miltefosine, Perifosine, CAL101, PX-866, LY294002,rapamycin, temsirolimus, everolimus, ridaforolimus, Alvocidib,Genistein, Selumetinib, AZD-6244, Vatalanib, P1446A-05, AG-024322,ZD1839, P276-00, GW572016 or a mixture thereof.

Yet further combination chemotherapies include, for example, alkylatingagents such as thiotepa and cyclosphosphamide; alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall; dynemicin, including dynemicin A; bisphosphonates, such asclodronate; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores, aclacinomysins,actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,pteropterin, trimetrexate; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens suchas calusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as mitotane, trilostane; folic acidreplenisher such as frolinic acid; aceglatone; aldophosphamideglycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;elformithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes such as cisplatin, oxaliplatin andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids such as retinoic acid;capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien,navelbine, farnesyl-protein tansferase inhibitors, transplatinum, andpharmaceutically acceptable salts, acids or derivatives of any of theabove. In certain embodiments, the compositions provided herein may beused in combination with gefitinib. In other embodiments, the presentembodiments may be practiced in combination with Gleevac (e.g., fromabout 400 to about 800 mg/day of Gleevac may be administered to apatient). In certain embodiments, one or more chemotherapeutic may beused in combination with the compositions provided herein.

b. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as y-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic composition and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

c. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, inconjunction with a TUSC2 therapy of the present embodiments. The generalapproach for combined therapy is discussed below. Generally, the tumorcell must bear some marker that is amenable to targeting, i.e., is notpresent on the majority of other cells. Many tumor markers exist and anyof these may be suitable for targeting in the context of the presentembodiments. Common tumor markers include carcinoembryonic antigen,prostate specific antigen, urinary tumor associated antigen, fetalantigen, tyrosinase (p9′7), gp68, TAG-72, HMFG, Sialyl Lewis Antigen,MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155.

d. Gene Therapy

In yet another embodiment, the secondary treatment is a gene therapy inwhich a therapeutic polynucleotide is administered before, after, or atthe same time as the therapeutic composition. Viral vectors for theexpression of a gene product are well known in the art, and include sucheukaryotic expression systems as adenoviruses, adeno-associated viruses,retroviruses, herpesviruses, lentiviruses, poxviruses including vacciniaviruses, and papiloma viruses, including SV40. Alternatively, theadministration of expression constructs can be accomplished with lipidbased vectors such as liposomes or DOTAP:cholesterol vesicles. All ofthese method are well known in the art (see, e.g. Sambrook et al., 1989;Ausubel et al., 1998; Ausubel, 1996).

Delivery of a vector encoding one of the following gene products willhave a combined anti-hyperproliferative effect on target tissues. Avariety of proteins are encompassed within the present embodiments, someof which are described below.

i. Inhibitors of Cellular Proliferation

As noted above, the tumor suppressor oncogenes function to inhibitexcessive cellular proliferation. The inactivation of these genesdestroys their inhibitory activity, resulting in unregulatedproliferation.

Genes that may be employed as secondary treatment in accordance with thepresent embodiments include p53, p16, Rb, APC, DCC, NF-1, NF-2, WT-1,MEN-I, MEN-II, zac1, p′73, VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p2′7,p27/p16 fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-1,TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fms, trk, ret, gsp, hst,abl, E1A, p300, genes involved in angiogenesis (e.g., VEGF, FGF,thrombospondin, BAI-1, GDAIF, or their receptors), MCC and other geneslisted in Table IV.

ii. Regulators of Programmed Cell Death

Apoptosis, or programmed cell death, is an essential process for normalembryonic development, maintaining homeostasis in adult tissues, andsuppressing carcinogenesis (Kerr et al., 1972). The Bcl-2 family ofproteins and ICE-like proteases have been demonstrated to be importantregulators and effectors of apoptosis in other systems. The Bcl-2protein, discovered in association with follicular lymphoma, plays aprominent role in controlling apoptosis and enhancing cell survival inresponse to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary andSklar, Proc. Nat'l. Acad. Sci. USA, 82(21):7439-43, 1985; Cleary et al.,1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). Theevolutionarily conserved Bcl-2 protein now is recognized to be a memberof a family of related proteins, which can be categorized as deathagonists or death antagonists.

Subsequent to its discovery, it was shown that Bcl-2 acts to suppresscell death triggered by a variety of stimuli. Also, it now is apparentthat there is a family of Bcl-2 cell death regulatory proteins whichshare in common structural and sequence homologies. These differentfamily members have been shown to either possess similar functions toBcl-2 (e.g., Bcl_(XL), Bcl_(W), Bcl_(S), Mcl-1, A1, Bfl-1) or CounteractBcl-2 Function and Promote Cell Death (e.g., Bax, Bak, Bik, Bim, Bid,Bad, Harakiri).

e. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatmentsprovided herein, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). It is further contemplated that the present embodiments may beused in conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

f. Anti-Inflammatory Agents

In certain aspects TUSC2 therapies are administered in conjuction withan anti-inflammatory agent. An anti-inflammatory agent is defined hereinto refer to an agent that is known or suspected to be of benefit in thetreatment or prevention of inflammation in a subject. Corticosteroidsare a major class of anti-inflammatory agent. The corticosteroids may beshort, medium, or long acting, and may be delivered in a variety ofmethods. A non-limiting list of corticosteroids contemplated in thepresent embodiments include the oral corticosteroids such as: cortisone,hydrocortisone, prednisone, and dexamethasone.

Another major class of anti-inflammatory agents are non-steroidalanti-inflammatory agents. Non-steroidal anti-inflammatory agents includea class of drugs used in the treatment of inflammation and pain. Theexact mode of action of this class of drugs is unknown. Examples ofmembers of this class of agents include, but are not limited to,ibuprofen, ketoprofen, flurbiprofen, nabumetone, piroxicam, naproxen,diclofenac, indomethacin, sulindac, tolmetin, etodolac, flufenamic acid,diflunisal, oxaprozin, rofecoxib, and celecoxib. One of ordinary skillin the art would be familiar with these agents. Included in thiscategory are salicylates and derivates of salicylates, such as acetylsalicylic acid, sodium salicylate, choline salicylate, choline magnesiumsalicylate and diflunisal.

Other anti-inflammatory agents include anti-rheumatic agents, such asgold salts (e.g., gold sodium thiomalate, aurothioglucose, andauranofin), anti-rheumatic agents (e.g., chloroquine,hydroxychloroquine, and penicillamine), antihistamines (e.g.,diphenhydramine, chlorpheniramine, clemastine, hydroxyzine, andtriprolidine), and immunosuppressive agents (e.g., methotrexate,mechlorethamine, cyclophosphamide, chlorambucil, cyclosporine, andazathioprine). Other immunosuppressive agent contemplated by the presentembodiments is tacrolimus and everolimus. Tacrolimus suppressesinterleukin-2 production associated with T-cell activation, inhibitsdifferentiation and proliferation of cytotoxic T cells. Today, it isrecognized worldwide as the cornerstone of immunosuppressant therapy.One of ordinary skill in the art would be familiar with these agents,and other members of this class of agents, as well as the mechanism ofactions of these agents and indications for use of these agents.

g. Other Agents

It is contemplated that other agents may be used in combination with thecompositions provided herein to improve the therapeutic efficacy oftreatment. These additional agents include immunomodulatory agents,agents that affect the upregulation of cell surface receptors and GAPjunctions, cytostatic and differentiation agents, inhibitors of celladhesion, or agents that increase the sensitivity of thehyperproliferative cells to apoptotic inducers. Immunomodulatory agentsinclude tumor necrosis factor; interferon alpha, beta, and gamma; IL-2and other cytokines; F42K and other cytokine analogs; or MIP-1,MIP-1beta, MCP-1, RANTES, and other chemokines. It is furthercontemplated that the upregulation of cell surface receptors or theirligands such as Fas/Fas ligand, DR4 or DR5/TRAIL would potentiate theapoptotic inducing abilities of the compositions provided herein byestablishment of an autocrine or paracrine effect on hyperproliferativecells. Increases intercellular signaling by elevating the number of GAPjunctions would increase the anti-hyperproliferative effects on theneighboring hyperproliferative cell population. In other embodiments,cytostatic or differentiation agents can be used in combination with thecompositions provided herein to improve the anti-hyperproliferativeefficacy of the treatments. Inhibitors of cell adhesion are contemplatedto improve the efficacy of the present invention. Examples of celladhesion inhibitors are focal adhesion kinase (FAKs) inhibitors andLovastatin. It is further contemplated that other agents that increasethe sensitivity of a hyperproliferative cell to apoptosis, such as theantibody c225, could be used in combination with the compositionsprovided herein to improve the treatment efficacy.

In certain embodiments, hormonal therapy may also be used in conjunctionwith the present embodiments or in combination with any other cancertherapy previously described. The use of hormones may be employed in thetreatment of certain cancers such as breast, prostate, ovarian, orcervical cancer to lower the level or block the effects of certainhormones such as testosterone or estrogen. This treatment is often usedin combination with at least one other cancer therapy as a treatmentoption or to reduce the risk of metastases.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsprovided herein. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the present embodiments, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the present embodiments.

Example 1 Clinical Study Design

Eligible patients were required to have histologically documentednon-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC) notcurable by standard therapies and previously treated with platinum-basedchemotherapy. The primary end point was assessment of DOTAP:chol-TUSC2toxicity during cycle 1 and determination of the maximum tolerated dose(MTD). Cycles consisted of a single intravenous infusion every 21 days.Secondary end points included TUSC2 plasmid expression in pretreatmentand 24 hour post treatment tumor specimens from subjects consenting totumor biopsies and tumor response. The presence of viable cancer cellsin the biopsied lesion was confirmed in all cases by histopathologicalexamination. Mandatory biopsies were explicitly precluded by regulatorycommittees at the local and federal level. Tumor response assessed bycomputed tomography (CT) scans was determined in accordance withstandard World Health Organization (WHO) criteria (Miller et al., 1981).This study was approved by the University of Texas MD AndersonInstitutional Review Board, the NIH Recombinant DNA Advisory Committee,and the FDA. All patients provided written informed consent prior toentry into the study.

Other eligibility criteria included: Eastern Cooperative Oncology Group(ECOG) performance status ≦1; adequate hematologic, hepatic, and renalfunction; prothrombin time and partial thromboplastin time ≦1.25 timesthe upper limit of normal; left ventricular ejection fraction >50%;forced expiratory volume in 1 second (FEV1) and diffusing capacity ofthe lung for carbon monoxide (DLCO) ≧40% of predicted; and negativehuman immunodeficiency virus serology test. Exclusion criteria included:prior gene therapy; brain metastases, unless treated, asymptomatic, andnot requiring steroid therapy; chemotherapy within 21 days beforeenrollment; radiation therapy within 30 days before enrollment;investigational therapies within 30 days before enrollment; activeinfection requiring antibiotic therapy; myocardial infarction or anginawithin 6 months before enrollment; and pregnancy or lactation.

A history and physical examination were performed before every cycle.Adverse events were assessed and laboratory tests performed prior toeach cycle and on days 2, 3, and 8. Laboratory tests included a completeblood count with differential, sodium, potassium, chloride, calcium,albumin, total protein, blood urea nitrogen, creatinine, alanineaminotransferase, aspartate aminotransferase, alkaline phosphatase,lactate dehydrogenase, and total bilirubin. Urinalysis andelectrocardiograms were obtained prior to each cycle.

The primary end point was assessment of DOTAP:chol-TUSC2 toxicity duringcycle 1 and determination of the maximum tolerated dose (MTD). Secondaryend points included tumor response and TUSC2 plasmid expression inpretreatment and 24 hour posttreatment tumor specimens from subjectsconsenting to tumor biopsies. DOTAP:chol-TUSC2 was administered atescalating doses as a 30 minute infusion in a peripheral vein in a totalvolume of 100 mL of 5% dextrose solution. Patients receivedDOTAP:chol-TUSC2 every 21 days for up to 6 treatments. After the ninthpatient was enrolled, the protocol was amended to requirediphenhydramine 50 mg orally or intravenously 30 minutes prior totreatment and dexamethasone 8 mg orally 24 and 12 hours beforetreatment, 20 mg intravenously 30 minutes prior to treatment, and 8 mgorally 12, 24, and 36 hours after treatment.

The initial starting dose (0.02 mg/kg) was selected based on toxicologystudies in non-human primates. This dose was one tenth the dose whichresulted in no deaths in non-human primates. After the sixth patient wasenrolled, the starting dose was amended to 0.01 mg/kg. Dose escalationwas based on a continuous reassessment method (CRM) which allows the MTDto be periodically re-estimated (O'Quigley et al., 1990). The MTD wasdefined as the highest dose level in which no more than 10% of patientsdevelop dose-limiting toxicity (DLT), defined as grade 3 non-hematologicor hematologic toxicity during cycle 1 judged by the investigator to berelated to DOTAP:chol-TUSC2. Patients entered at a given dose level werenot eligible for dose escalation or dose reduction. A cohort of 3patients was treated at each dose level. After treating 3 patients at agiven dose level, the information of whether the patients developed DLTwas used to compute the posterior probability of toxicity. Only toxicityduring cycle 1 was used to determine the next dose level. If noDOTAP:chol-TUSC2-related toxicities were observed in any prior patient,the subsequent dose level was increased by 100%. If only grade 1 or 2toxicities were observed, the subsequent dose level was increased by50%. If any DLT was observed, the CRM could lead to either escalation orreduction of dose levels. If DLT occurred and the CRM resulted in a doseescalation, the subsequent dose level was increased by 25%. Toxicity wasgraded according to the National Cancer Institute Common ToxicityCriteria, version 2.0. Tumor status was assessed at baseline and afterevery two cycles of therapy with computed tomography (CT) scans and/orpositron emission tomography (PET)/CT scans. Tumor response assessed bycomputed tomography (CT) scans was determined in accordance withstandard World Health Organization (WHO) criteria. 10 Additional detailson patient selection and assessment are provided in the SupplementaryMethods. Dr. J. Jack Lee designed the clinical trial and analyzed thedata. This study was approved by the University of Texas MD AndersonInstitutional Review Board, the NIH Recombinant DNA Advisory Committee,and the FDA. All patients provided written informed consent prior toentry into the study.

Example 2 FUS1/TUSC2 Expression Vector

This pLJ143/KGB2/FUS1 plasmid vector (FIG. 1A) includes a mammaliangene-expression cassette driven by a CMV minimum promoter with an E1enhancer at the 3′ end and a BGH-poly A signal sequence at the 5′ end toensure the efficient expression of the transgene in vivo. Thekanamycin-resistance gene was chosen as the selectable marker to avoiddevelopment of antibiotic-resistance in patients. A minimum pMB1 originof replication (ori) sequence is used to drive high-copy replication andproduction of the plasmid in the bacterial host strain DH5a. The plasmidbackbone is minimal to ensure a higher yield of plasmid DNA productionand a higher concentration of recombinant plasmid DNA per plasmid DNApreparation. The entire DNA sequence of the plasmid vector wasdetermined by automated DNA sequencing using the DNA Sequencing CoreFacility at the M.D. Anderson Cancer Center. The complete DNA sequenceof pLJ143/KGB2/FUS1 plasmid vector is provided as SEQ ID NO: 1. Specificelements of the vector are detailed below.

E1 Enhancer (bases 4-473): The E1 enhancer is a transcriptional enhancerfor adenoviral gene E1 protein and is derived from the adenoviralshuttle vector constructed by Grahm et al. E1 enhancer is used toenhance transcription of gene of interest under the control of CMVpromoter in mammalian cells.

CMV Promoter (bases 474-1171):

The CMV promoter is derived from the adenoviral shuttle vectorconstructed by Grahm et al. The CMV promoter is covered under U.S. Pat.Nos. 5,168,062 and 5,385,839, owned and licensed by the University ofIowa Research Foundation (Iowa City, Iowa 52242). The humancytomegalovirus (CMV) promoter has been cloned, sequenced, and used toconstruct a series of mammalian cell expression plasmid (Chapman et al.,1991). A high level of gene expression can be achieved under the controlof CMV promoter in mammalian cells.

BGH Polyadenylation Signal (Bases 1652-1877):

BGH polyadenylation signal sequence is derived from the adenoviralshuttle vector constructed by Grahm et al. The BGH polyadenylationsequence is covered under U.S. Pat. No. 5,122,458 and licensed byResearch Corporation Technologies (Tucson, Ariz.). Transcriptionaltermination by RNA polymerase III at the 3′ end of eukyrotic genesrequires two distinct cis-active elements, a functional poly (A) signaland a downstream transcription pause site. The BGH poly A signal hasbeen widely used as a transcription termination signal for mammaliangene expression in vitro and in vivo (Eggermont et al., 1993; Goodwin etal., 1992)

Kanamycin Resistance Gene (Bases 2049-2934):

The antibiotics Kanamycin resistance gene is derived from the pVAX1plasmid vector from Invitrogen (Carlsbad, Calif.). The kanamycinresistance gene is used as selective marker for plasmid production inbacterial E. coli. in the presence of antibiotics Kanamycin.

pMBJ Origin (Bases 2995-3734):

The high copy number plasmid pMB1 replication origin sequence is derivedfrom the pMG plasmid vector from Invivogen (San Diego, Calif.). Theminimal pMB1 origin is used to reduce plasmid size and drive a high copynumber replication of plasmid DNA in E. coli.

Example 3 Plasmid Preparation

The pLJ143/KGB2/FUS1 plasmid vector was produced under GMP conditions atthe Baylor College of Medicine Center for Cell and Gene Therapy(Houston, Tex.) and the Beckman Research Institute of the City of Hope(Duarte, Calif.).

A 1 mL vial of the pLJ143 master cell bank stock was asepticallyinoculated into 500 mL sterile Terrific Broth with 1.6% Glycerol(Teknova: 1.2% Tryptone, 2.4% yeast extract, 1.6% glycerol. lx phosphatebuffer) supplemented with Kanamycin (Sigma) and grown overnight (15-18hours) at 37° C. This was then used to inoculate 20 L of Terrific Brothin a New Brunswick scientific BioFlo IV 4500 fermentor, operating at 37°C., 250-300 rpm, 20-30% CO₂. Cells are harvested by centrifugation,washed once (1 mL buffer per g wet cell paste) with Alkaline LysisSolution I (Teknova: 50 mM Glucose, 25 mM Tris-HCl, pH 8.0, 10 mM EDTA,pH 8.0, sterile solution) and frozen at −80° C.

The cell pastes were removed from −80° C. storage and thawed in a 4° C.refrigerator overnight. The cell paste was mixed with Alkaline LysisSolution I at 8 ml per gram of wet cell paste. The pLJ143 suspension wasthen mixed 1:1 (v/v) with Alkaline Lysis Solution II (Teknova: 200 mMNaOH, 1.0% SDS, sterile solution). After mixing, the material wasallowed to lyse at room temperature between 8 and 10 minutes. Twovolumes of Alkaline Lysis Solution III (Teknova: 3M potassium acetate,1.18M formic acid, pH 5.5, sterile solution) were then added with thelysate, and mixed on ice to ensure complete neutralization andprecipitation of host cell proteins, genomic host cell DNA and SDS. Theneutralized cell lysate was clarified using a bucket centrifuge at 4000rpm for 30 minutes at 4° C. The supernatant was decanted and clarifiedthrough a 1.2 mM PP2 filter.

This four step purification process does not require RNase enzyme,organic solvents, detergents, precipitants or animal derived components.The entire process is controlled with an Aekta Purifier (AmershamBioscience) and Unicorn Software (Amersham Bioscience). All columns andpacking material are from Amersham Bioscience. All column preparationand storage is as follows:

CIP: 0.5 M NaOH, 25° C. for I hour contact time

Depyrogenation: 100 ppm sodium hypochlorite pH 10, then 0.1-0.5N NaOH,pH13

Storage: 20% ethanol (aqueous solution)

Preparation for Use (Sanitization): Cell culture grade water (USPharmacopia), then primed with applicable buffer

Step 1: Concentration Using Hollow Fiber Filter (HFF)

The clarified lysate was first concentrated approximately 10-fold andequilibrated with using a 300,000 kDa nominal molecular weight cut-off(NMWCO) A/G Technology hollow fiber filter (HFF). The HFF was flushedwith 4-L of Alkaline Lysis Solution III (3M potassium acetate, 1.18Mformic acid, pH 5.5, sterile solution) and pooled with the concentratedlysate. A final volume of approximately 2-L is recovered, filtered witha 0.45 μm filter, and stored at 4° C. until the next step.

Step 2: Size Exclusion

RNA removal and buffer exchange by group separation using Sepharose 6Fast Flow with BPG size exclusion column. UF Concentrate is applied tothe column in batches of 0.3 column volumes (CV) to change the buffer toBuffer A (2M (NH₄)SO₄, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0).Simultaneously this procedure also removes RNA and other contaminants.The void fractions are stored at 4° C. and then pooled for the nextstep.

Step 3: Selective Capture of Supercoiled Plasmid DNA by ThiophilicAromatic Adsorption Chromatography.

Supercoiled plasmid DNA is separated from open circular plasmid DNA andremaining contaminants such as residual genomic DNA and RNA. The pooledvoid fraction (from Step 2) is subsequently applied on the XK50 Affinitycolumn packed with PlasmidSelect and equilibrated in the same Buffer A.The column is washed and supercoiled plasmid DNA is eluted with Buffer B(1.4M NaCl, 2.0 M (NH₄)SO₄, 10 mM EDTA, 100 mM Tris-HCl, pH7.0.).Fractions are stored at 4° C. prior to next step. Fractions are pooledfor step 4. then diluted with four volumes of water for the next step.

Step 4: Polishing and Concentration with SOURCE 30Q

Endotoxins are further removed and at the same time, the supercoiledplasmid DNA preparation is concentrated by ion exchange chromatography.The fraction (from step 3) containing supercoiled plasmid DNA is dilutedwith 4 volumes of pharmaceutical grade water and loaded on a XK26 ionexchange column packed with SOURCE 30Q. The column is equilibratedBuffer C (0.4 M NaCl, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0) and elutedwith a linear gradient, Buffer D (1.0 M NaCl, 10 mM EDTA, 100 mMTris-HCl, pH 7.0). The fractions are then pooled and filtered through a0.22 μm filter.

PlasmidSelect is the key protocol component since it interactsdifferentially with nucleic acids by thiophilic aromatic adsorption inthe presence of water structuring salts. This enables thetopoisomere-selective purification of native supercoiled plasmid DNA andremoval of damaged, nicked or open circular DNA by simple adjustment ofchromatographic conditions. A group separation for removal of RNA priorto application on the column optimizes the capacity of PlasmidSelect forbinding of the supercoiled form of plasmids. Furthermore, groupseparation with Sepharose 6 Fast Flow greatly reduces the risk ofprecipitation during addition of ammonium sulfate and limits thevariation in initial salt concentration that can influence selectivity,thus giving the process considerable robustness.

Ethanol precipitation was used to concentrate the pLJ143 to 5 mg/ml. A3.0 M sterile NaCl solution was used to increase the NaCl concentrationof the pLJ143 solution to 0.15M. Ethanol was added into the pLJ143solution in a 2:1 ratio to give a final ethanol concentration of 67%.The pLJ143 suspension was stored at −20° C. overnight to allow forcomplete precipitation. The next morning, the pLJ143 was recovered bycentrifugation. The pLJ143 pellets were further washed with 70% ethanoland allowed to air dry aseptically in a laminar flow hood forapproximately one hour. Dried pellets were frozen at −80° C. untilpurification of wet paste from all fermentation runs was complete. ThepLJ143 was then reconstituted at 5 mg/ml in sterile endotoxin, RNAse,DNAse free water. All work is performed in a class 100 biosafetycabinet.

Product was filled into 1.2 ml crimp cap glass vials with semi-automateddispensing pipette in a class 100 biosafety cabinet. The fill volume iseither 0.3 ml, 0.5 ml, or 1.0 ml. The target plasmid DNA concentrationwas 5 mg/mL. The final product is stored at −80° C. Plasmid purity testsand quality control standards are shown in Table 1 below.

TABLE 1 Plasmid quality control specifications. Character- istics TESTSpecifications Appear- Clear, Colorless ance pH pH meter 5.5 ≦ pH ≧ 8.0DNA DNA Homogeneity ≧90% Appropriate form DNA Identity Supercoiled DNA≧95% Restriction Map Restriction Digestion pattern Identical to thereference Bio-activity by cell trans- Positive for Transgene Expres-fection and Western- sion blotting Purity A260/280 Ratio 1.7-2.0A260/230 Ratio 2.0-2.2 Protein Contamination <10 μg/mg DNA Host E. coliGenomic <1% (w/w) of total detectable DNA Contamination nucleic acid byqPCR Residual RNA Contamina- <5% (w/w) of total detectable tion nucleicacid (0.5 φg load) by gel Residual Isopropanol and by GC analysisethanol Residual Antibiotics Undetectable (<3.0 φg/ml) (kanamycin)Ammonia ≦1 mg/mL of final formulated plasmid DNA Sulfate ≦1 mg/mL ofFinal Formulated Plasmid DNA Endotoxin <5 EU/mg DNA by LAL AssaySterility Bacterial (CFR) Negative Fungal Negative In vitro AdventitiousVirus Negative Concen- O.D. At 260 nm 1 to 5 mg/mL/vial ± 0.5% trationper vial Biologic Transfection in H1299 cells Protein expression byWestern Activity Blot

Example 4 Nanoparticle Preparation

DOTAP GMP grade was purchased from Avanti Polar Lipids, Inc. (Alabaster,Ala.) and cholesterol GMP grade was purchased from Sigma-Aldrich (St.Louis, Mo.). A ratio of 20 mM DOTAP:18 mM cholesterol was used forpreparation of the nanoparticles. The reagents were mixed and the drylipids dissolved in purified GMP grade chloroform. A Buchi rotaryevaporator was used to form a dry lipid film. Further drying wasperformed under a vacuum in a Labconco Freeze dry system. The film wasresuspended in sterile 5% dextrose in water. After sonication thefollowing day under aseptic conditions the lipids are sequentiallyextruded through a series of sterile Whatman filters from 1 μm to 0.1 μmin pore size.

The diluted plasmid DNA and diluted nanoparticle stock were mixed inequal volumes to a final concentration of 4 mM DOTAP and 0.5 mg/ml ofDNA. Prior to treatment the assigned dose was diluted in 100 ml D5W. Anegative gram stain was required prior to treatment.

Example 5 Therapy Protocol and Results

Thirty-one patients were enrolled in the study at a single institution.Patient characteristics are described in Table 2.

TABLE 2 Baseline Characteristics of Patients Character- No. of Patients(%) istic (n = 31) Median age, years (range) 60 (43-76) Sex Male 16(51.6%) Female 15 (48.4%) ECOG performance status  0 4 (12.9%)  1 27(87.1%) Histology Adenocarcinoma 11 (35.5%) Bronchioalveolar carcinoma 1(3.2%) Squamous cell carcinoma 3 (9.7%) Non-small cell carcinoma, NOS 11(35.5%) Small cell carcinoma 5 (16.1%) Prior Therapy Chemotherapy 31(100%) Radiotherapy 14 (45.2%) Surgery 11 (35.5%) Prior Chemotherapyregimens  1 9 (29%)  2 9 (29%) >2 13 (41.9%) Number of doses received  18 (26%)  2 19 (61%) >3 4 (13%) Abbreviations: ECOG, Eastern CooperativeOncology Group; NOS, not otherwise specified

A total of 74 cycles of DOTAP:chol-TUSC2 were administered, with amedian of 2 cycles (range, 1 to 12 cycles) per patient. Patients weretreated at 6 dose levels ranging from 0.01 to 0.09 mg/kg. The doseescalation scheme, including number of patients, number of cycles, DLTs,and grade 2 toxicities judged to be related to DOTAP:chol-TUSC2 arelisted in Table 3.

TABLE 3 Dose-Escalation Scheme Dose Grade 2 Cohort level No. of No. ofNo. patients toxicity No. (mg/kg) Patients cycles with DLT (No.patients)  1 0.02 3 9 0 Fever (1)  2 0.03 3 6 0 0  3¹ 0.01 3 4 2; G3fever Fever (1) (n = 2), G3 hypotension (n = 1)  4* 0.01 3 9 0 0  5*0.02 3 6 0 0  6* 0.04 3 6 0 0  7* 0.06 3 6 0 ALT (1), neuropathy (n = 1) 8* 0.09 3 5 1; G3 hypo- Fever (1) phosphatemia  9* 0.06 3 16 0 Hypo-phosphatemia (1), nausea (1), myalgia (1) 10* 0.06 3 5 1; G3 hypo- Fever(1), phosphatemia myalgia (1), hypo- phosphatemia (1), 11 0.06 1 2 0 0Abbreviations: G3, grade 3; ALT, alanine aminotransferase elevation¹This cohort did not receive dexamethasone or diphenhydraminepremedications *Cohorts used to determine maximum tolerated dose (MTD)

The first patient in cohort 1 (receiving 0.02 mg/kg) developed grade 2fever within 3 hours of the DOTAP:chol-TUSC2 infusion. The subsequentpatients in cohorts 1 and 2 were given dexamethasone and diphenhydramineprior to receiving DOTAP:chol-TUSC2, and no grade 1 or higher toxiciteswere observed. However, after discussions with the FDA, it was mandatedthat the next patient cohort receive DOTAP:chol-TUSC2 at a lower doselevel of 0.01 mg/kg without dexamethasone or diphenhydraminepremedication. All three patients developed grade 2 or 3 fever and onepatient developed grade 3 hypotension. The FDA then allowed the protocolto be amended to require dexamethasone and diphenhydraminepremedications beginning with the next cohort (patient 10), starting ata dose level of 0.01 mg/kg. Due to this amendment, it was decided not touse the toxicity data from the first nine patients for MTDdetermination, and a subgroup of 21 patients enrolled between Sep. 28,2006 and Oct. 29, 2009 were used to determine the final MTD.

The only subsequent DLTs observed were grade 3 hypophosphatemia in twopatients with one at 0.06 mg/kg and another at 0.09 mg/kg. In both casesthe patients had either grade 1 or 2 fevers and the hypophosphatemia wasan incidental laboratory finding. The MTD was determined to be 0.06mg/kg. As listed in Table 2, grade 2 toxicities included myalgias,hypophosphatemia, fever, nausea, and transaminase elevation.

Peripheral Blood Mononuclear Cell (PBMC) Cryo-Preservation andFluorescent Activated Cell Sorter Analysis (FACS)

Patient blood samples were collected in capped glass tubes containingficoll at room temperature (RT). Blood samples were centrifuged at 3500rpm at RT for 30 minutes in a swing-out rotor. Separated plasma andlymphocytes were collected separately in a centrifuge tubes. An equalvolume of PBS was added to the lymphocyte-containing tube andcentrifuged at 900×g at RT for 10 min. After centrifugation, thesupernatant was removed. The cell pellets were washed again with thesame volume of PBS as the first wash. The cell suspension wascentrifuged at 700×g for 10 minutes, and the supernatant was removed.Cell concentrations were determined and adjusted to a finalconcentration of 5×106 cells/mL with cell-freezing medium containing 10%of DMSO and 90% fetal bovine serum. Eight hundred uL aliquots of5×106/mL lymphocytes were transferred into cryogenic vials. ThePBMC-containing cryogenic vials were stored in a −80° C. freezer for 48h and then transferred to a liquid nitrogen freezer.

Frozen PBMC were thawed immediately in a 37° C. water bath, then washedwith 10 ml of RPMI1640 with 10% FBS. The cells were then lysed with 1×BD FACS Lysing Solution (BD Biosciences, San Jose Calif.) for 10 minutesat room temperature. The cells were centrifuged at 400×g for 10 minutes,followed by treatment with 1×FACS Permeabilizing Solution 2 (BDBiosciences, San Jose, Calif.) for 10 minutes at room temperature. Thecells were then rinsed with PBS containing 1% FBS and centrifuged for 10minutes at 400×g and re-suspended in 400 μL of PBS with 1% FBS. Aliquotswere made in the required number of BD Falcon 5 mL polystyrene tubes.Antibodies (BD, Franklin Lakes, N.J.) were then added to each tubeaccording to the table listed below under the fluorescent dye in boldletters:

FITC PE PerCP APC IgG1 IgG1 CD14 IgG1 TNF-a IL-6 CD14 IL-15 IL-1b IFN-gCD14 IL-8 CD14

The cells were incubated with antibodies for 30 minutes at roomtemperature protected from light, washed with PBS containing 1% FBS,re-suspended in 250 uL of PBS with 1% paraformaldehyde, and analyzed by6-color flow cytometry (LSRII, BD). The cytokine data was analyzed usingFlowJo software (Tree Star, Inc., Ashland, Oreg.).

Results of these studies showed that intracellular levels of TNF-a,IL-15, IL-6, IL1b, IFNg, and IL-8 in peripheral blood monocytes andlymphocytes remained unchanged 24 hours after treatment (FIG. 11A-B).

Antibodies to Single and Double Stranded DNA

Serum antibodies to single and double stranded DNA were determined by anELISA assay performed at the Mayo Clinic Department of LaboratoryMedicine and Pathology, Rochester, Minn. For single stranded DNAantibodies a value of <69 U/ml is considered negative for antibodydetection. For double stranded DNA antibodies a value of <1 isconsidered negative for antibody detection.

Results showed that antibodies to single and double stranded DNA werenot detected 14 months after completion of 12 cycles of therapy inpatient 26.

Example 6 TUSC2 RNA Expression

All Specimens were Blinded for Patient Identity, for ClinicalInformation and for specimen timing (pre- vs post-treatment) during allstudies. Ectopic expression of the TUSC2 gene in patient biopsy sampleswas analyzed using a TaqMan™ based quantitative real time reversetranscriptase-polymerase chain reaction (RT-PCR) (Applied Biosystems,Foster City, Calif.) that enables quantification of gene expression froma limited amount of starting material as detailed below.

RNA was isolated using RNeasy™ minikit from Qiagen (Valencia, Calif.)following the manufacturer's instructions. The fine-needle biopsytissues that were immediately fixed in RNAlater (Ambion, Austin, Tex.)were washed once with cold PBS and then the total RNAs were isolatedwith (reagent and methods). The quality of the purified RNA was analyzedusing an Agilent 2100 Nano Bioanalyzer (Agilent Technologies, SantaClara Calif.). Reverse transcription was done using a High Capacity cDNAReverse Transcription Kit (Applied Biosystems, Foster City, Calif.) withMultiScribe Reverse Transcriptase for two hours at 37° C. in a thermalcycler according to the manufacturer's instructions. The PCR reactionwas setup with 10 ml of 2×TaqMan™ gene expression master mix containingthe polymerase, buffer and dNTPs, 1 ml of 20×TaqMan™ gene expressionassay solution containing primers, probe, and 5 ml of cDNA template, and4 ml of sterile distilled water. The primers and probes used werespecific to the exogenous TUSC2 transcripts expressed through theplasmid gene expression cassette (Forward primer: 5′ GGA CCT GCA GCC CAAGCT 3′ (SEQ ID NO: 3) and Reverse primer: 5′ GCC CAT GTC AAG CCG AAT T3′ (SEQ ID NO: 4), and TaqMan™ probe: 6-FAM-CGA GCT CGG ATC CAC TAG TCCAGT GTG-TAMRA; SEQ ID NO: 5) to avoid detection of endogenous TUSC2mRNA. PCR analysis was performed using a 7500 Real-Time PCR System(Applied Biosystems, Foster City, Calif.) and run with an absolutequantification mode with a standard curve. The DNA amount values werethen used for the calculation of TUSC2 copy numbers using the Universityof Rhode Island's website available on the world wide web aturi.edu/research/gsc/resources/cndna.html (URI Genomics & SequencingCenter Calculator for determining the number of copies of a template).

TUSC2 transgene RNA expression by RT-PCR was not detected inpre-treatment biopsies (Table 4). Five of six post-treatment biopsiesshowed expression of the TUSC2 transgene. In a seventh patient (PatientNo. 31), TUSC2 mRNA was detected by RT-PCR transgene specific primersincluded in the qRT Profiler Apoptosis PCR Array and was detected onlyin the post-treatment sample (see below). Expression was not detected inpre- and post-treatment peripheral blood lymphocytes collected at thetime of the biopsies.

TABLE 4 Real Time RT-PCR detection of TUSC2 gene expression in patients.Tumor Tumor TUSC2 Lymphocy Pa- TUSC2 Gene Copy to TUSC2 tient Dose Siteof Expression Number Gene Num- (mg/ Tumor (pg/ug (copies/ Expression berkg) Biopsy Treatment tissue) ug tissue) (pg/μl) 1 0.02 Lung Pre- 0 0 NA¹treatment Lung Post-  2.0 × 10⁻⁵ ± 4.44 NA treatment 2.20 × 10⁻¹⁰ 7 0.01Lung Pre- 0 0 NA treatment Lung Post-  3.6 × 10⁻⁶ ± 0.89 NA treatment 9.1 × 10⁻⁷ 13 0.02 Lung Pre- 0 0 NA treatment Lung Post-  3.0 × 10⁻⁵ ±6.22 NA treatment 1.71 × 10⁻⁸ 20 0.06 Liver Pre- 0 0 0 treatment LiverPost- 0 0 0 treatment 24 0.09 Subcu- Pre- 0 0 0 taneous treatment noduleSubcu- Post-  8.0 × 10⁻⁶ ± 1.90 0 taneous treatment 2.33 × 10⁻⁸ nodule25 0.06 Lung Pre- 0 0 0 treatment Lung Post-  4.0 × 10⁻⁵ ± 8.76 0treatment 1.66 × 10⁻⁹ ¹Specimens not available

Example 7 TUSC2 Protein Expression

Anti-TUSC2 antibody was used to detect TUSC2 protein expression in pre-and post-treatment lung tumor biopsies from patients 13, 26 and 31 (FIG.2B). Specifically, Duolink kits from Olink Biosciences (Uppsala, Sweden)were used. These kits are based on PLA technology and the rolling circleamplification (RCA) reaction wherein a pair of oligonucleotide labeledsecondary antibodies (PLA probes) generates a signal only when the twoPLA probes have bound in close proximity, either to the same primaryantibody or two primary antibodies that have bound to the sample inclose proximity. The signal from each detected pair of PLA probes isvisualized as an individual fluorescent spot. Signals can be quantified(counted) and assigned to a specific subcellular location based onmicroscopy images.

The samples were incubated with primary antibodies that bind to theprotein(s) to be detected. Secondary antibodies conjugated witholigonucleotides (PLA probe MINUS and PLA probe PLUS) were then added tothe reaction and incubated. The ligation solution, consisting of twooligonucleotides and ligase, is added and the oligonucleotides hybridizeto the two PLA probes and join to form a closed circle if they are inclose proximity. The amplification solution, consisting of nucleotidesand fluorescently labeled oligonucleotides, was added together withpolymerase. The oligonucleotide arm of one of the PLA probes acts as aprimer for a rolling-circle amplification (RCA) reaction using theligated circle as a template, generating a concatemeric (repeatedsequence) product. The fluorescently labeled oligonucleotides thenhybridizes to the RCA product. The signal was visible as a distinctfluorescent spot that can be analyzed by fluorescence microscopy. Inorder to detect posttreatment TUSC2 protein expression, a singleantibody (TUSC2) raised in rabbits and oligo probes (plus and Minus)with rabbit secondary antibodies were used. In situ PLA was performed asper the recommendations of the manufacturer with minor modifications andalso including appropriate controls. The experiments were carried out ina blinded setting. Patient biopsy tissues preserved in RNAlater™ werewashed in 50 ml of cold PBS for 30 minutes at 4° C. before using OCT toprepare frozen blocks to cut slides. The slides with were then fixedwith 4% paraformaldehyde and permeabilized with methanol for 20 minuteseach. The tissues were blocked for 30 min at 37° C. in a humidifiedchamber with the blocking buffer provided in the kit and later incubatedwith anti-rabbit TUSC2 primary antibody overnight at 4° C. The followingday, the primary antibodies were washed and tissues incubated witholigo-linked secondary antibodies (anti-rabbit PLA probes plus andminus). Hybridization, ligation, amplification and detection were thenperformed according to the manufacturer's instructions. For non-specificcontrol, rabbit HA tag antibodies were used in the place of TUSC2antibody. For competition experiments, the synthetic oligopeptide(GASGSKARGLWPFASAA; SEQ ID NO: 2) derived from the N-terminal amino-acidsequence of the TUSC2 protein that was used to develop anti-TUSC2polyclonal antibody in rabbits was used (Ito et al., 2004).

The number of in situ proximity ligation signals was counted using thefreeware software Blobfinder (available on the world wide web atcb.uu.se/˜amin/BlobFinder). Nuclei were visualized by DAPI staining andused for cell count. The protein expression level was quantified bycounting all signals (fluorescent spots) obtained from one image dividedby the number of cells in the image, to derive the average signals/cell.Background subtraction was them applied with the pre-treatment samples.

Results of these studies are shown in FIG. 2A-B and demonstrated thatboth post-treatment biopsies showed a high level of TUSC2 protein withabsence of TUSC2 protein on the paired pre-treatment biopsies. Anon-specific control antibody showed only background staining.Pre-incubation of the TUSC2 antibody with the specific TUSC2 peptideused to immunize for antibody production, but not a non-specificpeptide, was able to significantly reduce TUSC2 fluorescence in thepost-treatment biopsies

Example 8 Effects on the Apoptosis Pathway

The expression of major genes in apoptosis signaling pathways inpretreatment and posttreatment needle biopsy specimens were quantifiedusing a qRT Profiler Apoptosis PCR Array with RT Nano PreAmp-mediatedcDNA synthesis (SA Biosciences, Frederick, Md.). The quantitativeapoptotic gene expression data were analyzed as detailed below andthrough the use of Ingenuity Pathway Analysis (IPA) Ingenuity Systems,(available on the world wide web at ingenuity.com).

For gene expression profiling experiments, the total RNAs were isolatedfrom patient fine needle biopsies using Trizol (Invitrogen, Carlsbad,Calif.) reagent and purified using a RT2 qPCR-Grade RNA isolation kitfrom SA Biosciences (Frederick, Md.) according to the manufacturer'sinstructions. The purified RNA was then used to synthesize cDNA usingRT2 Nano PreAmp cDNA Synthesis Kit from SA Biosciences (Frederick, Md.).This cDNA kit also involved pre-amplification of the cDNA targettemplates. The preamplified cDNA was applied onto a RT2 ProfilerApoptosis PCR array (SA Biosciences) for qPCR analysis using an ABI 7500real-time PCR instrument (Applied Biosystems, Foster City, Calif.)according to the manufacturers' instructions. The expression level ofthe mRNA of each gene in the patient after treatment withDOTAP:chol-TUSC2 was normalized using the expression levels ofhouse-keeping genes B2M, HPRT1, RPL13A, GAPDH, and ACTB. For dataanalysis, the comparative Ct method was used wherein the relativechanges in gene expression were calculated using the ΔΔCt (thresholdcycle) method. This method first subtracts the ct (threshold cyclenumber) of the gene-average ct of the five housekeeping genes on thearray (B2M HPRT1, RPL13A, GAPDH and ACTB) to normalize to the RNAamount. Finally, the ΔΔCt was calculated as the difference between thenormalized average ct of each gene on the array after DOTAP:chol-TUSC2treatment and the normalized average ct of the pre-treatment controlsample. This ΔΔCt was then raised to the power of 2 to calculate therelative fold-change of gene expression after-treatment compared topre-treatment. Genes that differed from pretreatment controls by morethan two fold were considered significant and changes of gene expressionlevels larger than three-fold were shown as a scatter plot.

The expression of major genes in apoptosis signaling pathway in tumorfine needle biopsies from human lung cancer patients before and aftersystemic treatment with DOTAP:chol-TUSC2 nanoparticles were quantifiedusing a qRT Profiler Apoptosis PCR Array with RT Nano PreAmp-mediatedcDNA synthesis (SA Biosciences, Frederick, Md.). The quantitativeapoptotic gene expression data were analyzed through the use ofIngenuity Pathway Analysis (IPA) Ingenuity Systems, (see, e.g., theworld wide web at ingenuity.com). For the network and canonical pathwayanalysis, the quantitative PCR data set containing gene identifiers andcorresponding expression fold change values was uploaded into theapplication. Each identifier was mapped to its corresponding object inIngenuity's Knowledge Base (IKB). An expression fold change(posttreatment/pretreatment) cutoff of 3 was set to identify moleculeswhose expression was significantly differentially regulated. Thesemolecules, called Network Eligible molecules, were overlaid onto aglobal molecular network developed from information contained in IKB.Networks of Network Eligible Molecules were then algorithmicallygenerated based on their direct or indirect connectivity. The Networkmolecules associated with biological functions in IKB were consideredfor analysis. Right-tailed Fisher's exact test was used to calculate ap-value determining the probability that each biological functionassigned to a given network is due to chance alone. Molecules from thedata set that met the above gene expression fold changes cutoff werealso considered for the canonical pathway analysis. The significance ofthe association between the data set and the canonical pathway wasmeasured by a ratio of the total number of molecules from the data setthat map to the pathway to the total number of molecules that map to thecanonical pathway in IKB. A Fisher's exact test was used to calculate ap-value determining the probability that the association between thegenes in the dataset and the canonical pathway is explained by chancealone.

Significant differences in gene expression were detected by an apoptosismultiplex array between a pre and post-treatment biopsy from patient No.31 whose tumor biopsies showed high levels of TUSC2 mRNA and proteinpost-treatment (FIG. 1B). The changes in gene expression and canonicalapoptosis pathways in TUSC2-mediated apoptosis are graphicallyrepresented by FIG. 1C. Analysis methods are detailed below.

Example 9 Response and Survival

Twenty-three patients received two or more doses. Five patients achievedstable disease (range 2.6 to 10.8 months, median 5.0, 95% CI 2.0-7.6)and all other patients progressed. Two patients had reductions inprimary tumor size of 14% and 26%. One patient with stable disease(patient 26) had evidence of a durable metabolic response on positronemission tomography (PET) imaging (FIG. 3) and received 12 cycles oftherapy. The response was documented with PET scans performed after thesecond, fourth (FIG. 2), and sixth doses, all showing decreasedmetabolic activity with no changes in size or number of metastases by CTimaging. This patient remains alive on subsequent therapy 14 monthsafter the final treatment with DOTAP:chol-TUSC2. Median survival for allpatients was 8.3 months (95% CI 6.0-10.5 months,) and mean survival timewas 13.2 months (95% CI 8.9-7.5 months) with a range of 2 to 21+months).

Example 10 Predicting Clinical Benefit

Formalin fixed paraffin embedded (FFPE) pretreatment tumor samplesobtained at initial diagnosis were available from 10 patients forassessment of baseline TUSC2 protein expression and AI. Only FFPE tissuecould be used for this assay. All pre and posttreatment biopsiesobtained specifically for this protocol were preserved in RNAlater(e.g., Patients 13 and 31) and could not be used for IHC.

TUSC2 Protein Expression

Formalin-fixed and paraffin-embedded (FFPE) tissue histology sections (5μm thick) were baked overnight at 56°, deparaffinized, hydrated.Heat-induced epitope retrieval was performed in a DAKO antigen retrievalbath (10 mmol/L of sodium citrate, pH 6.0) at 121° C. for 30 seconds and90° C. for 10 seconds in a decloaking chamber (Biocare, Concord,Calif.), followed by a 30-min cool down. Peroxide blocking was done with3% H₂O₂ in methanol at room temperature for 15 min, followed by 10%bovine serum albumin in TBS-t for 30 min. The slides were incubated withprimary antibody at 1:400 dilution for 65 min at room temperature. Afterwashing with TBS-t, incubation with biotin-labeled secondary antibodyfor 30 min followed. The samples were incubated with a 1:40 solution ofstreptavidin-peroxidase for 30 min. The staining was then developed with0.05% 3′,3-diaminobenzidine tetrahydrochloride prepared in 0.05 mol/L ofTris buffer at pH 7.6 containing 0.024% H₂O₂ and then counterstainedwith hematoxylin. Formalin-fixed and paraffin-embedded lung tissues withnormal bronchial epithelia were used as a positive control. For anegative control, the same specimens used for the positive controls wereused, replacing the primary antibody with TBS-t. TUSC2 immunostainingwas detected in the cytoplasm of normal epithelium and tumor cells.Immunohistochemical expression was quantified by two independentpathologists (M. I. Nunez and I. I. Wistuba) using a four-valueintensity score (0, 1+, 2+, and 3+) and the percentage of the reactivityextent. A consensus value on both intensity and extension was reached bythe two independent observers. A final consensual score was obtained bymultiplying both intensity and extension values (range, 0-300).

TdT-Mediated dUTP Nick End Labeling (TUNEL) Assay and Apoptotic Index

FFPE tissue sections were stained using the DeadEnd™ Colorimetric TUNELSystem (Cat G7130, Promega, Madison, Wis.) according to technicalmanufacture recommendation. The negative controls were performedomitting the rTdT enzyme in the TUNEL reaction mixture. The positivecontrols were performed treating the tissues with DNase I enzyme (Cat#M6101, Promega, Madison, Wis.) prior to the reaction mixture. 10high-powered fields (×400) per case were assessed (at least 1000 cells).The apoptotic index (AI) was the total number of TUNEL positive cellsper 1000 cells counted.

Results of these studies showed that TUSC2 protein expression inpretreatment FFPE tumor biopsies was low in most of the pretreatmentbiopsies with only two samples exceeding the level noted in normalbronchial epithelium (FIG. 4). The level of pretreatment TUSC2 proteinexpression did not correlate with clinical benefit. TUNEL staining wasalso performed in 10 pretreatment biopsies and an apoptotic index (AI)was calculated as detailed above. High levels of AI (>10%) wereassociated with achieving a minor response or stable disease while thosewith an AI of ≦10% all had progressive disease (FIG. 5).

Example 11 TSC2 Therapy in Combination with EGFR-Targeted Therapy

To assess cooperative effects of tumor growth data between FUS-1 (FUS1)and Erlotinib (Erlo) a Bayesian Bootstrapping analysis approach wasused. The Pr(min(μ_(F), μ_(E))<μ_(C)|data) was calculated i.e., theposterior probability that the minimum of the two posterior mean colonyformation for FUS-1 alone, μ_(F), or Erlotinib alone, μ_(E), is lessthan the mean posterior colony formation for the combination μ_(C). Thisprobability calculates the likelihood that average colony formation inthe combination arm will be less than the minimum of the two singleagent arms. Cooperative effects are shown if this posterior probabilityis large. Thus, the probability of cooperative effect ranges from 0.0 to1.0 where 0 means no chance of a true cooperative effect given the dataobserved while 1 means 100% certainty of a cooperative effect given thedata observed. The Statistical software S-PLUS 8.0 were used for all theanalyses.

In the studies presented here all cell lines, including H1975 cells,which have two EGFR mutations (L858R/T790M), and doses of erlotinibshowed almost near certainty of a cooperative effect (See, FIGS. 6-10and Tables 5-8). The probability of cooperative effectiveness calculatedfrom the results of each of the studies is provided below:

For 1299 cells: FUS1+Erlotinib (1.0 μs) (Probability of CooperativeEffect=1.00); FUS1+Erlotinib (2.3 μs) (Probability of CooperativeEffect=1.00). See Table 5; FIG. 6.

For H322 cells: FUS1+Erlotinib (1.0 μg) (Probability of CooperativeEffect=1.0); FUS1+Erlotinib (2.3 μs) (Probability of CooperativeEffect=1.0). See Table 6; FIG. 7.

For A549 cells: FUS1+Erlotinib (1.0 μg) (Probability of CooperativeEffect=0.9981); FUS1+Erlotinib (2.3 μg) (Probability of CooperativeEffect=1.00). See Table 7; FIG. 8.

For H460 cells: FUS1+Erlotinib (1.0 μg) (Probability of CooperativeEffect=0.9874); FUS1+Erlotinib (2.3 μg) (Probability of CooperativeEffect=1.0). See Table 8; FIG. 9.

For H1975 cells: FUS1+Erlotinib (1.0 μg) (Probability of CooperativeEffect=1.0); FUS1+Erlotinib (2.3 μg) (Probability of CooperativeEffect=1). See FIG. 10.

TABLE 5 Fus1 and Erlotinib Combine Treatment Effect on Colony Formationof H1299 Cells. pc301 + pc301 + EV + Fus1 + Fus1 + Fus1 + Group pc301PBS1 2.3 PBS EV + 1 EV + 2.3 PBS 1 2.3 1 40355.0 31957.0 24425.0 15796.018287.0 9216.0 12651.0 6212.0 4315.0 2 39639.0 25107.0 18192.0 18653.015301.0 8082.0 10095.0 5307.0 4633.0 3 47131.0 32817.0 21246.0 17517.017010.0 7128.0 9988.0 7396.0 4070.0 average 42375 29960 21288 1732216866 8142 10911 6305 4339 SD 4134 4225 3117 1438 1498 1045 1508 1048282 CV %  9.8% 14.1% 14.6%  8.3% 8.9% 12.8% 13.8% 16.6% 6.5% P value of0.013 0.009 0.406 0.010 0.027 0.009 Ttest erlo diffent dose 0.014 0.0040.062 EV vs FUS1 0.7231 0.0009 0.0060 or Erlo Fus1 + 1 vs 0.0006 0.0122Fus1 or Erlo Fus1 + 2.3 0.0037 0.0018 vs Fus1 or Erlo normalized  100%  71%   50%   41%  40%   19%   26%   15%  10% on pc301 normalized  100% 97%   47%   63%   36%  25% on EV normalized  8.3% 8.6%  6.0%  8.7% 6.0% 1.6% % SD

TABLE 6 Fus1 and Erlotinib Combine Treatment Effect on Colony Formationof H322 Cells pc301 + pc301 + EV + Fus1 + Fus1 + Fus1 + Group pc301PBS 12.3 PBS EV + 1 EV + 2.3 PBS 1 2.3 1 57216 44761 28610 46711 43621 2029323947 11162 3203 2 65399 37701 28943 46887 36971 16469 19851 11259 36533 55676 35079 27080 53930 35119 17511 18660 8947 2782 average 5943039180 28211 49176 38570 18091 20819 10456 3213 SD 5226 5008 994 41184471 1977 2773 1308 436 CV %  8.8% 12.8% 3.5%  8.4% 11.6% 10.9% 13.3%12.5% 13.6% P value of 0.022 0.003 0.073 0.004 0.007 0.004 Ttest erlodiffent dose 0.026 0.003 0.003 EV vs FUS1 0.0391 0.0003 0.0006 or ErloFus1 + 1 vs 0.0005 0.0042 Fus1 or Erlo Fus1 + 2.3 0.0002 0.0004 vs Fus1or Erlo normalized  100%   66%  47%  83%   65%   30%   35%   18%   5% onpc301 normalized  100%   78%   37%   42%   21%   7% on EV normalized 8.4%  9.1%  4.0%  5.6%  2.7%  0.9% % SD

TABLE 7 Fus1 and Erlotinib Combine Treatment Effect on Colony Formationof A549 Cells. pc301 + pc301 + EV + Fus1 + Fus1 + Group pc301PBS 1 2.3PBS EV + 1 EV + 2.3 PBS Fus1 + 1 2.3 1 4147 3779 1625 2714 3083 24841358 932 511 2 6208 2436 1803 3147 2538 1910 1732 1279 435 3 6586 33931651 2716 2334 1738 1308 1358 460 average 5647 3203 1693 2859 2652 20441466 1190 469 SD 1313 691 96 249 387 391 232 227 39 CV % 23.2% 21.6%5.7%  8.7% 14.6% 19.1% 15.8% 19.0% 8.3% P value of 0.073 0.016 0.2780.057 0.116 0.011 Ttest erlo diffent dose 0.040 0.000 0.021 EV vs FUS10.4791 0.0382 0.0021 or Erlo Fus1 + 1 vs 0.0049 0.2138 Fus1 or ErloFus1 + 2.3 0.0023 0.0018 vs Fus1 or Erlo normalized  100%   57%  30%  51%   47%   36%   26%   21%   8% on pc301 normalized  100%   93%   71%  51%   42%  16% on EV normalized  8.7% 13.5% 13.7%  8.1%  7.9% 1.4% %SD

TABLE 8 Fus1 and Erlotinib Combine Treatment Effect on Colony Formationof H460 Cells. Group EV + PBS EV + 1 EV + 2.3 Fus1 + PBS Fus1 + 1 Fus1 +2.3 1 2117 1216 750 1564 1158 330 2 2179 1393 968 1751 1322 261 3 21061470 858 2018 1094 289 average 2134 1360 859 1778 1191 293 SD 39 130 109228 118 35 CV %  1.8% 9.6% 12.7% 12.8% 9.9% 11.8% P value of 0.005 0.0010.037 0.005 T-test on erlo diffent dose 0.006 0.004 EV vs FUS1 0.00060.00004 0.056 or Erlo Fus1 + 1 vs 0.1719 0.0167 Fus1 or Erlo Fus1 + 2.30.0010 0.0004 vs Fus1 or Erlo normalized  100%  64%   40%   83%  56%  14% on EV normalized  1.8% 6.1%  5.1% 10.7% 5.5%  1.6% % SD

The use of FUS1 expression to enhance the effectiveness of gefitinib andovercome gefitinib resistance was also explored in human NSCLC.Re-expression of wild-type FUS1 by FUS1-nanoparticle-mediated genetransfer into FUS1-deficient and gefitinib-resistant NSCLC cell linesH1299, H322, H358, and H460 cells that have a wild-type EGFRsignificantly (P<0.001) sensitized their response to gefitinib treatmentand synergistically induced apoptosis in vitro and in an H322 orthotopiclung cancer mouse model (FIG. 12, Note that these studies included theK-ras mutant cell line H460 which is significant in that patients withK-ras mutant tumors are in general unresponsive to EGFR TKIs).Supra-additive induction of apoptosis was seen with the combination ofnanoparticle FUS1 and concentrations of gefitinib similar tosteady-state serum concentrations achievable with oral dosing. Tounderstand the mechanism of gefitinib-induced resistance, agefitinib-resistant HCC827GR NSCLC cell line (IC₅₀=16 μM) wasestablished by selecting against gefitinib from the parental HCC827cells that contain an activating deletion mutation of the EGFR gene andare extremely sensitive to gefitinib treatment (IC_(50=0.016) uM). Nosecondary mutations in the EGFR gene in the HCC827GR cells was found,but these cells registered a significantly elevated level ofphosphorylated AKT protein. Combination treatment withFUS1-nanoparticles and gefitinib at a dose level of IC₁₀ significantlyre-sensitized the cells to gefitinib, as demonstrated by synergisticallyenhanced growth inhibition and apoptosis. FUS1 nanoparticle treatmentalone or with gefitinib markedly inactivated EGFR and AKT, as shown bydecreased phosphorylation levels of both proteins on Western blots,compared with either agent alone (FIG. 12D). Cleavage of caspase-3,caspase-9, and PARP was also significantly induced by the combination ofFUS1 and gefitinib in HCC872GR and other gefitinib-resistant NSCLCcells. The combination of FUS1 and erlotinib induced similar levels oftumor cell growth inhibition, apoptosis induction, and inactivation ofoncogenic PTKs as those observed in NSCLC cells treated by a combinationof FUS1 and gefitinib (FIG. 12A-D).

Example 12 In Vivo Assessment of TSC2 Therapy in Combination withEGFR-Targeted Therapy

The cooperative interaction between erlotinib and FUS1 nanoparticles wasconfirmed in vivo using a lung colony formation metastases model innu/nu mice with A549 human lung cancer cells injected in the tail vein.Following injection mice were treated with FUS1 nanoparticles anderlitinib and various controls (FIG. 13). The greatest reduction in lungcolonies occurred with the FUS1 nanoparticle/erlotinib combination (90%reduction) which was significantly reduced compared to all controlgroups (p<0.0005).

These studies along with those in Example 11 showed that a combinationtreatment of FUS1 nanoparticles and gefitinib or erlotinib can promote asynergistic tumor cell killing and overcome drug-induced resistance bysimultaneously inactivating the EGFR and the AKT signaling pathways andby inducing apoptosis in resistant cells with wild-type EGFR.

Example 13 Bystander Effect of FUS1-Nanoparticle in NSCLC

Many currently available gene transfer protocols and techniques arecapable of transducing only a fraction of tumor cells in vivo, and thus,relying on a bystander effect (killing of non-transduced cells byproducts of transduced cells) to achieve clinically-meaningfultherapeutic efficacy. For example, bystander effects have been observedin cancer gene therapy by adenoviral or retroviral vector-mediated genetransfer of tumor suppressor genes such as p53 and TRAIL and for suicidegene HSV-TK in cancer cells. These bystander effects are induced throughvarious mechanisms including intercellular communication, interaction ofcell surface receptors and ligands, secretion of cytotoxic or apoptoticmetabolites and peptides, and activation of anticancer cytokine cascadesand the immune response. To test whether ectopic expression of FUS1 intumor cells can cause neighboring cell killing by triggering the releaseof cytotoxic soluble factors, conditioned medium (CM) was collected fromFUS1-transduced H1299 cells. CM was collected after 48 h in cell cultureeither containing or free of bovine fetal serum (BFS) and concentrated2-5 fold by lyophilization. The CMs from untransduced (PBS), or emptyvector (EV), and myristoylation-deficient mutant FUS1(mt-FUS1)-transduced cells were used as controls. A marked inhibition oftumor cell growth (FIG. 14A) and induction of apoptosis (FIG. 14B) weredetected in H1299 cells treated by concentrated CMs fromwt-FUS1-transduced cells compared with those of controls. In addition,distinct soluble protein/peptide species were clearly detected in theserum-free wt-FUS1-CM on protein mass spectra by a ProteinChiparray-based SELDI-TOF-MS analysis (FIG. 14C), compared to those ofcontrol CMs, suggesting release of specific soluble peptides. To furthertest the potential bystander effects of FUS1 on lung cancer cells, thewt-FUS1-nanoparticles-transfected H1299 cells were used as effectorcells and mixed them with the Ad-GFP-transduced H1299 target cells,which do not express FUS1, at a ratio of 1:1. The empty vector(EV)-nanoparticle-transfected H1299 effectors were used as the control.The mixed cells were then seeded into a 6-well plate and cultured for 48hr. The dead/apoptotic cells were labeled by PI staining and analyzed byflow cytometry to determine the extent of cell death and apoptosis inboth effector and target (GFP) cells. An increased population ofdead/apoptotic cells was detected in the GFP-expressing target cellsmixed with wt-FUS1-transfected H1299 effecter cells, compared with thatof target cells mixed with EV-transfected effectors (FIG. 15). Thiseffect is comparable to that seen with a secreted protein such as TRAIL.These preliminary data support the presence of bystander effects inducedby FUS1-nanoparticle-mediated gene transfer in lung cancer cells.

Example 14 Preclinical Animal Studies with FUS1-Nanoparticles

Murine Studies

The mouse LD₁₀ for a single intravenous dose of DOTAP:Cholesterol-Fus1liposome complex was determined from a series of experiments. For eachexperiment, C3H strain mice (4 to 6 weeks old, estimated total bloodvolume 1 ml) were injected over a period of approximately 3 minutes. Thedoses ranged from 50 to 150 mcg of DOTAP:Cholesterol-Fus1 liposomecomplex, and the total injection volume ranged from 100 to 300microliters. The results of the dose-escalation study in mice aresummarized below in Table 9.

TABLE 9 DOTAP: Chol-Fus1 dose escalation in mice DOTAP: Chol- TotalInjection Number of Fus1 dose number of volumn deaths (mcg) mice (ml)(%) 50 8 100 0 60 8 120 0 70 8 140 0 80 8 160 0 90 8 180 0 100 23 200  2(8.6%) 110 18 220 0 120 18 240  1 (5.6%) 130 18 260 7 (39%) 150 23 300 7(30%)

The LD₁₀ for a single intravenous injection in mice was conservativelyestimated to be 100 micrograms. Of significance, the drug was infusedover approximately 3 minutes, and the injection volumes ranged from 100to 300 microliters, or the equivalent of 10 to 30% of the animals' totalblood volume. This rapid rate of infusion would never be used in humans,and the relationship of the rapid infusion rate to the observed animaltoxicity remains unclear.

Autopsies were obtained on all animals that died secondary to acutetoxicity. Pathological examination of the brain, heart, lungs, spleen,liver, gastrointestinal tract, and kidneys were performed by anattending veterinary pathologist. The pathology findings are summarizedbelow in Table 10.

TABLE 10 Pathology in DOTAP: Chol-Fus1 treated mice Dose Number of (mcg)autopsies Pathology findings (number of animals) 100 2 Lymphoid tissueand spleen, necrosis, apoptosis, and atrophy, moderate (2) Multifocalliver degeneration and necrosis, mild (1) Acute liver necrosis, mild (1)120 1 Lymphoid tissue, spleen, and GALT necrosis, apopto- sis, andatrophy, moderate (1) Acute liver necrosis, moderate (1) Malignantlymphoma, kidney (1) Glomerulonephritis (1) 130 7 Lymphoid tissue andspleen, necrosis, apoptosis, and atrophy, mild (1), moderate (6) Acuteliver necrosis, mild (3), moderate (3), severe (1) Multifocal myocardialdegeneration, necrosis, and mineralization, moderate (2), severe (1)Acute tubular necrosis, kidney, minimal (1) Lung granuloma/foreignbodies (1) Intestinal crypt epithelial acute necrosis, mild (1) 150 7Lymphoid tissue and spleen, necrosis, apoptosis, and atrophy, mild (3),moderate (4) Acute liver necrosis, mild (4), moderate (1), severe (2)Multifocal myocardial degeneration, necrosis, and mineralization, mild(1), moderate (2) Acute tubular necrosis, kidney, mild (1) Multiplesubacute to chronic kidney infarcts (1) Spleen red pulp myeloidhyperplasia (1) Spleen sinus histiocyte marked hyperplasia (1)Intestinal crypt epithelial acute necrosis, mild (1) Note: Multifocalmyocardial degeneration, necrosis, and mineralization are most likelyincidental findings observed in control C3H mice (ref. Vargas, K J,Stephens, L C, Clifford, C B, et al. Dystrophic Cardiac Calcinosis inC3H/HeN Mice. Lab Anim Sci, 46: 572-575, 1996.) Minimal = 1+, 5-10%;Mild = 2+, 10-20%; Moderate = 3+, 20-50%: Severe = 4+, >50%.

GLP Toxicology Studies

The objective of this study was to determine single dose toxicology ofDOTAP:Chol/fus1 in preparation for Phase I studies. The non-toxic doseand dose-limiting toxicity for C3H/HeNCR mice were determined. The studycontained three control groups: D5W (vehicle), 4 mM DOTAP:Chol (highestdose of lipid), and 70 μg DNA (highest dose of fus1 plasmid). The studyalso contained three experimental groups: 70 μg DNA,DOTAP:Chol, 40 μgDNA,DOTAP:Chol and 10 μg DNA, DOTAP:Chol. Each group contained 15 mice(8 female and 7 male). Acute (0-72 hours), subacute (14 days) andchronic (6 weeks) toxicity were evaluated. At 3 and 14 days and at 6weeks, five mice per group were euthanized. For each mouse, an attemptwas made to collect urine for analysis for CBC and serum chemistries.Necropsies were performed and histopathological analysis done on allmice, including those that died during the study. This study wasconducted in an AAALAC accredited facility (2000).

All mice in the three control groups (D5W, 4 mM DOTAP:Chol, and 70micrograms DNA alone) and in the experimental group receiving 10micrograms DNA, DOTAP:Chol were observed to be normal at all observationtime points.

Mice in the experimental group receiving 40 micrograms DNA, DOTAP:Cholappeared normal at the end of the 4 hours post-injection observationperiod. When observed later that day at approximately 7 hours postinjection 14/15 mice were squinting and appeared to be lethargic. Onefemale mouse was very weak, trembling and sat hunched with her eyesclosed. She was euthanized and sent to necropsy at that time. On day onepost injection (PI), all mice had decreased activity levels and the eyesappeared to be swollen. On day two PI, all mice appeared to havereturned to normal activity levels and general appearance. One femalemouse had an area of necrosis involving approximately 20% of one pinnaat this time point, but otherwise appeared normal. The damaged pinna wasinterpreted to be the result of trauma. All mice were thereafter normalat all observation time points. In summary, one female mouse becamemoribund on day zero and was euthanized.

Mice in the experimental group receiving 70 micrograms DNA, DOTAP:Cholappeared normal at the end of the 4 hours post-injection observationperiod. When observed later that day at approximately 7 hours postinjection, all mice were squinting and appeared to be lethargic. On dayone PI, one female mouse died. Three male mice and one female mouse werefound to be moribund and were euthanized and necropsied. One femalemouse was reported to have a swollen face. This mouse and the remainingmice in the group all appeared to have decreased activity levels andabnormal appearance at day one PI. On day two PI, the female mouse thathad the swollen face on day one PI was found to be moribund and waseuthanized and necropsied. Another female mouse was found dead on daytwo PI. The remaining mice had decreased or slightly decreased activitylevels and some were squinting. On day three PI, 2/8 remaining miceappeared normal, while 6/8 still had decreased activity levels andabnormal general appearance. From day four PI and thereafter, all miceappeared normal at all observation time points. In summary, two femalemice died. Three male and two female mice were found moribund and wereeuthanized.

Non-Human Primate Toxicology

Ten (10) cynomolgus monkeys (Macaca fascicularis) were used in thestudy. Six experimental animals (three male and three female) wereinjected with DOTAP:Chol/Fus 1 complex on Day 1 and Day 21 of the study.Four control animals (two male and two female) were injected withDOTAP:Cholesterol alone on Day 1 and Day 21 of the study. At days 46-52the animals were necropsied, blood was collected for hematology andchemistries, and organs were collected for histopathological analysis.

Significant gross and microscopic lesions were found in 1/10 monkeys onprotocol. This animal received 1 dose of 0.6 mg/kg DNA, DOTAP: Chol(high dose) and died within 18-20 hours. Lesions in this monkey weremost likely treatment related. A second monkey that received the highdose of DNA, DOTAP: Chol had changes in a lymph node. The significanceof these minimal changes is not known. Equivocal lesions were found inthe femoral bone marrow of two low dose (0.2 mg/kg DNA, DOTAP: Chol)monkeys. The latter may be incidental findings, but were not seen inother protocol animals. No significant gross or microscopic lesions werefound in the remaining six animals that received either DOTAP: Chol onlyor 0.2 mg/kg DNA, DOTAP: Chol.

Example 15 Enhancement of Anti-Tumor Activity of MK2206 in Human LungCancer Cells by Tumor Suppressor Gene FUS1

Studies were undertaken to investigate whether FUS1 nanoparticles cansensitize lung cancer cells to chemotherapeutic agents such as MK2206.First preliminary studies were performed to determine the DCtransfection efficiency in various lung cancer cell lines. Results ofthese studies are shown below in Tables 11 and 12.

TABLE 11 Cell lines with high DC transfection efficiency Cell GFP line(%) H2882 53.9 H1395 52.9 H2450 51.4 H358 46.1 H1299 40.2 H1171 37.8H2887 34.6 H661 33.1 H522 30.8 Calu-1 25 H1650 24.8 H322 24.7 HCC82723.6 HCC366 22.4

TABLE 12 Cell lines with low DC transfection efficiency Cell GFP line(%) H196 17 H460 11.46 H1944 11.4 H1703 11.3 H1355 9.03 H1648 8.9 Calu-68.6 H1993 8.4 H1975 7.97 Calu-3 7.74 HCC193 7.53 H2052 6.01 H515 6 H20093.51 H838 2.88 H2935 1.83 H1792 1.67 H157 1 H3122 0.88 H226 0.56 H14370.44 H125 0.36

Next the effect of FUS1 nanoparticle treatment alone was assessed in anarray of lung cancer cell lines. As shown in FIG. 16, FUS1 waseffectively expressed in the HCC366, H322, A549 and H2887 cell lines.FUS1 expression resulted in a consistent (but not significant) decreasein cell viability in all cell lines (bottom panel).

Single drug treatment with AKT inhibitor MK2206 was also assessed in awide range of lung cancer cell lines. The effective IC₅₀ on the variouscells are shown in FIG. 17. Cell lines indicated by arrows (H322, A549,H2887 and HCC386) were subjected to further analysis. First, each of thecell lines was treated with FUS1 nanoparticles or empty vector atincreasing concentrations of MK2206. Results shown in FIG. 18 showsynergistic cell killing mediated by the combination of FUS1 and thekinase inhibitor. The effect of the combined therapy was especiallyevident in H2887, H322 and HCC366 cells. Next, the ability of combinedFUS1/MK2206 treatment to inhibit colony formation was studied in thecell lines. Graphs shown in FIG. 19 demonstrate that the combination ofMK2206 and FUS1 was significantly more effective than either treatmentalone at inhibiting colony formation. Thus, FUS1 treatment is able tosensitize cancer cells to the effects of kinase inhibitors such as theAKT inhibitor MK2206. This effect was quantified relative to eachstudied cell line below in Table 13.

Additional colony formation assays in both H322 and H1299 cellsdemonstrated that TUSC2 nanoparticles synergistically inhibited colonyformation in the cancer cells when combined with the EGFR-targetedtherapeutic afatinib (FIG. 26A-B). In these studies, afatinib showedeven greater effect in combination with TUSC2 than similarconcentrations of erlotinib combined with TUSC2. Still further studiesindicated that dasatinib has enhanced anti-cancer activity when used inconjunction with TUSC2 nanoparticles.

TABLE 13 Fold decrease in IC₅₀ of MK2206 when combined withFUS1-nanoparticles and gene mutation status IC₅₀ IC₅₀ (MK2206 (MK2206 +Fold Cell line alone) FUS1) reduction kras Braf EGFC PIK3CA LKB1 H32220.39 1.24 16.4 wt wt wt wt mutant HCC366 18.4 2.17 8.5 — — — — mutantH2887 16.53 1.28 12.9 — — — — — A549 2.86 0.56 5.1 mutant wt wt wtmutant

Further studies were undertaken to evaluate the ability of FUS1 andMK2206 treatment to induce apoptosis. Cells were treated with the twoagents, or each individually, stained by propidium iodide (PI) andanalyzed by flow cytometry. Results of these studies are shown in thehistograms of FIG. 20. In the case of each cell line, combined FUS1 andMK2206 treatment resulted in significantly more apoptotic cells ascompared to either agent alone (indicated by the horizontal bar in thehistograms).

To better determine the mechanism for synergistic FUS1/MK2206 effect,treated cells were subjected to an immunoblots to assess thephosphorylation status of cell signaling molecules. As shown in FIG. 21,FUS1 alone resulted in an increased in phosphorylated AMPK (p-AMPK), buthad little effect on the level of phosphorylated AKT (p-AKT). On theother hand, the addition of MK2206 significantly reduced phosphorylatedAKT levels, while a robust increase in phosphorylated AMPK (mediated byFUS1) was maintained. Indeed, the role of AMPK signaling inFUS1-mediated cell killing was confirmed by the fact that treatment ofcells with a AMPK-targeted siRNA partially protected the cells from theeffects of FUS1/MK2206 treatment (see, e.g., FIG. 22). Likewise, aninhibitor of AMPK activity (Compound C) was also able to partiallyprotect cancer cells from FUS1/MK2206-mediated killing (FIG. 23). Thus,FUS1-increased sensitivity to MK2206 is associated with thedown-regulation of AKT and mTOR phosphorylation and up-regulation ofAMPK phosphorylation. In view of these studies a proposed FUS/MK2206signaling pathway is provided as FIG. 25.

The in vivo effectiveness of combination FUS1 and MK2206 treatment wasfurther assessed using a mouse xenograft model. For these studies H322cells were transplanted into mice and the explanted cells allowed togrow in vivo. Tumor mass was assessed at various time points in thepresence of FUS1 therapy, MK2206 therapy or the combination of the two.In all cases expression of FUS1 and activity of MK2206 (as evidenced byreduced p-AKT expression) was histologically evaluated in samples fromthe mice. As shown in FIG. 24, results of these studies showed thatcombined FUS1 and MK2206 therapy was far more effective than eithertreatment alone at inhibiting tumor growth in the animals.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method for treating a subject having a cancer,comprising administering to the subject an effective amount of a TUSC2therapy, an epidermal growth factor receptor (EGFR) inhibitor and ananti-inflammatory agent to treat the cancer.
 2. The method of claim 1,wherein the TUSC2 therapy is administered after the epidermal growthfactor receptor (EGFR) inhibitor or the anti-inflammatory agent.
 3. Themethod of claim 1, wherein the TUSC2 therapy is administered before oressentially simultaneously with the epidermal growth factor receptor(EGFR) inhibitor or the anti-inflammatory agent.
 4. The method of claim1, wherein the cancer was determined to express an EGFR.
 5. The methodof claim 1, wherein the TUSC2 therapy comprises administration of aTUSC2 expression vector.
 6. The method of claim 5, wherein the TUSC2expression vector is plasmid DNA.
 7. The method of claim 5, wherein theTUSC2 expression vector is provided in a liposome.
 8. The method ofclaim 7, wherein the liposome is a DOTAP:cholesterol liposome.
 9. Themethod of claim 1, wherein the TUSC2 therapy comprises administration ofa TUSC2 polypeptide.
 10. The method of claim 9, wherein the TUSC2polypeptide is myristoylated.
 11. The method of claim 9, wherein theTUSC2 polypeptide is comprised in a nanoparticle.
 12. The method ofclaim 1, wherein the EGFR inhibitor is a tyrosine kinase inhibitor. 13.The method of claim 1, wherein the EGFR inhibitor is an EGFR bindingantibody.
 14. The method of claim 1, wherein the EGFR inhibitor iserlotinib, gefitinib, cetuximab, matuzumab, panitumumab, AEE788;CI-1033, HKI-272, HKI-357 or EKB-569.
 15. The method of claim 14,wherein the EGFR inhibitor is erlotinib.
 16. The method of claim 1,wherein the anti-inflammatory agent is an anti-rheumatic agent,non-steroidal anti-inflammatory agent or a gold salt.
 17. The method ofclaim 16, wherein the gold salt is auranofin, gold sodium thiomalate oraurothioglucose.
 18. The method of claim 17, wherein theanti-inflammatory agent is auranofin.
 19. The method of claim 1, furthercomprising administering a further anti-cancer therapy to the subject.20. The method of claim 19, wherein the second anti-cancer therapy ischemotherapy, radiotherapy, gene therapy, surgery, hormonal therapy,anti-angiogenic therapy or cytokine therapy.
 21. The method of claim 1,wherein the cancer is lung cancer.
 22. The method of claim 21, whereinthe lung cancer is non-small cell lung cancer.
 23. The method of claim21, wherein the lung cancer is a metastatic lung cancer.
 24. The methodof claim 1, wherein the cancer is EGFR inhibitor or chemotherapyresistant.
 25. A method for treating a subject having a cancer,comprising administering to the subject a TUSC2 therapy, erlotinib andauranofin in a therapeutically effective amount to treat the cancer. 26.A pharmaceutical composition comprising a TUSC2 therapeutic, erlotinib,and auranofin in pharmaceutically acceptable formulation.